Olimpus FLUOVIEW FV500 User Manual Download Page 313

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APPLIED OPERATIONS

/Image Analysis

2-203

Page

The magnification or scrolling of the graph can be canceled by

dragging the left button of the mouse from the bottom left to the right of

the magnified graph.

When the mouse pointer is placed on a graph line while the Ctrl or

Alt key is held depressed, the coordinates can be displayed.

The displayed data can be applied to other applications.

See section 2-10, “Transferring Data to Another Application” for

details.

TIP

Summary of Contents for FLUOVIEW FV500

Page 1: ... operation This user s manual is composed of two volumes including SYSTEM OVERVIEW OPERATION INSTRUCTIONS MAINTENANCE and TROUBLESHOOTING Together with this manual please also read the User s manual FLUOVIEW FV500 and the instruction manual of the microscope in order to understand overall operation methods To ensure the safety operation of laser system we recommend you to study the manual of each ...

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Page 3: ...ited 2 The information described in this manual may be subject to change without notice Registered Trademarks Microsoft Microsoft Windows Excel for Windows are registered trademarks of Microsoft Corporation Other brand names and product names are trademarks or registered trademarks of their respective owners ...

Page 4: ...es of warning labels SPECIFICATIONS Another manual Describes the specifications of the FLUOVIEW system SYSTEM OVERVIEW This manual Describes the outline of the FLUOVIEW system OPERATION INSTRUCTIONS This manual Describes the operation procedures of the FLUOVIEW system for example methods for image acquisition and various image processing MAINTENANCE This manual Describes maintenance of the FLUOVIE...

Page 5: ...ialog box list box or check box is enclosed inside square brackets OK button Open File button The name of a button in a panel or dialog box is enclosed inside angled brackets Notation of Mouse Operations Notation Description clicking Action of pressing then immediately releasing the mouse button double clicking Action of clicking the mouse button twice in quick succession dragging Action of moving...

Page 6: ...ion keys Generic names given to the and keys Notation of system specific terms Notation Description XY observation Other observations Refers to observation with XY scanning The same principle also applies to other observations such as XZ Xt XYZ XYt and XYZt Note that some of the panels and dialog boxes shown in this manual are not the precise reproductions of the originals Some windows are resized...

Page 7: ...This volume describes the outline of the FLUOVIEW FV500 system Please read this volume so that you can understand the system before use On This Volume S SY YS ST TE EM M O OV VE ER RV VI IE EW W ...

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Page 9: ...tion 1 4 1 4 1 System Component Units and Their Functions 1 4 1 4 2 Block Diagram 1 6 1 5 Software Functional Configuration 1 9 1 5 1 Function Panel and Display Panel 1 9 1 5 2 Panel Structure of the Software 1 10 1 5 3 Icons Executed by Dragging Dropping 1 11 1 5 4 Identification of Images Depending on the Observation Methods 1 12 ...

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Page 11: ...ctor and outputs the image of the specimen on an image monitor The confocal optics place a confocal aperture at a position which is optically conjugate with the focusing position i e confocal plane to eliminate light from parts other than those for the focusing position As a result the part corresponding to the eliminated light is darkened in the image making it possible to slice a thick tissue sp...

Page 12: ...n be acquired simultaneously 6 The scan unit can be used in common with an upright or inverted microscope 7 The confocal aperture is continuously variable and the optimum confocal aperture for each objective is set automatically 8 High speed scanning acquires 4 images per second 512 x 512 pixels 9 Sequential scan for acquiring images without crossover 10 In addition to raster scanning scanning mod...

Page 13: ... 1 3 Page 1 3 Light Path Diagram Emission DM turrets 1 to 3 Pinholes 1 to 4 Confocal lenses 1 to 4 Emission filter turret Mirror To IR laser unit To UV laser unit To VIS laser combiner Mirrors DM X Y galvano Mirrors Excitation DM turret ...

Page 14: ... the optical fiber cable Control unit Unit for driving the scanning unit laser combiner X motor and transmitted light detector Image monitor Video monitor for displaying laser scanning images control panel etc Transmitted light unit Unit incorporating a 100 W halogen lamp bulb to provide illumination for transmitted light observation Connected with the microscope through an optical fiber cable Ref...

Page 15: ...SYSTEM OVERVIEW System Configuration 1 5 Page Photo 1 2 Combination with IX Microscope Upright microscope exclusively designed for FLUOVIEW FV500 ...

Page 16: ...Dsub9pin Dsub9pin Dsub9pin IR LASER POWER SUPPLY 2m 3m Mercury burner U RFL T BH2 RFL T3 Reflected Fluorescence Light Power Supply Unit CHILLER FV5 LUUV FV5 LDUIR REMOTE SW 3 1m 10 2 2 9m 10 1 7m 1 2 9m 1 1 2 6 2 9m Square 3 pin HeCd LASER POWER SUPPLY 1 8m Kr LASER SIROCCO FAN Duct 2 2m UV Ar LASER POWER SUPPLY 2 2 9m 10 2m 7 2 9m U PS combination with AX only FV5 LUHECD 3 3m 2 2 9m 1 2 9m Circul...

Page 17: ... SUPPLY 2m 3m Mercury burner U RFL T BH2 RFL T3 Reflected Fluorescence Light Power Supply Unit CHILLER FV5 LUUV FV5 LDUIR REMOTE SW 3 1m 10 2 2 9m 10 1 7m 1 2 9m 1 1 2 6 2 9m Square 3 pin HeCd LASER POWER SUPPLY 1 8m Kr LASER SIROCCO FAN Duct 2 2m UV Ar LASER POWER SUPPLY 2 2 9m 10 2m 7 2 9m U PS combination with AX only FV5 LUHECD 3 3m 2 2 9m 1 2 9m Circular 8 pin Dsub25pin Dsub15pin Dsub15pin Ds...

Page 18: ... OFF UV laser and IR laser are optional For connection to PC PC SYSTEM 50 pin For connection to FV5 PSU PC SYSTEM 50 pin For switching signals output from the left connectors VIS1 and VIS2 are for invisible laser UV is for UV laser IR is for IR laser and Blanking is for switching laser ON OFF UV laser and IR laser are optional VIS1 VIS2 UV IR VIS1VIS2 UV IR Blanking Event output signal 1 TLL Event...

Page 19: ...xecuted just like when using a system notebook or file folder 1 5 1 Function Panel and Display Panel The FLUOVIEW software is organized by two kinds of panels the function panels and display panel The function panels include the Acquire File I O Tile Process Analyze and Visualize panels The display panel shows either the Live panel or the panel image loaded from a file filename panel In this manua...

Page 20: ...uisition File I O Tile Process Math Filters Histogram Logical Analyze Single Saves loads and deletes images Changes the image display method Processes the acquired images Performs mathematical operations between images Filters images Changes the image contrast Performs logical operations between images Analyzes image data Obtains the intensity values intensity distribution length area and average ...

Page 21: ...ragging Dropping This software selects image files and observation methods dye name by means of dragging dropping This allows simple selection based on an intuitive operation of selecting an icon image file or observation method dragging it to the desired position and dropping it there ...

Page 22: ...the Icon field in the Files list box in the File I O panel or during dragging of an image file Use these icons to identify the observation methods used in image acquisition In all observation modes the icons for 3 or more channels are identical Image Icon Significance XZ observation XZ observation 2 channel mode Xt observation Xt observation 2 channel mode XZT observation XZT observation 2 channel...

Page 23: ...system Getting Started FLUOVIEW contains information on the basic operation flow until acquisition of XY images APPLIED OPERATIONS provides detailed operating procedures of the system Please read this volume so that you can understand the system before use On This Volume ...

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Page 25: ...ination with Inverted Microscope BX50WI 1 33 1 2 4 3 Combination with Upright Microscope AX 1 35 1 2 4 4 Combination with Upright Microscope AX70A 1 37 1 2 4 5 Combination with Inverted Microscope IX 1 39 1 2 4 6 Combination of IXBP Bottom Port 1 41 1 2 5 Selecting the Dyeing Method 1 43 1 2 6 Selecting the Filters 1 46 1 2 7 Selecting the ND Filters 1 48 1 2 8 Setting the Observation Condition 1 ...

Page 26: ...1 Image Acquisition Procedure Section A 2 3 2 1 2 Image Acquisition Procedure in an Observation Mode Section B 2 4 2 1 3 Examples of Operation Procedures 2 5 2 2 Image Acquisition 2 7 2 2 1 Image Acquisition in XY Observation Mode 2 8 2 2 1 1 Configuring the Microscope 2 9 2 2 1 2 Setting the Filters 2 15 2 2 1 3 Setting the ND Filters 2 17 2 2 1 4 Setting the Observation Condition 2 18 2 2 1 5 Ac...

Page 27: ...86 2 2 11 1 Making REX Mask File 2 87 2 2 11 2 Example of FRAP experiment 2 92 2 3 Saving Opening and Shredding Images 2 101 2 3 1 Saving Images 2 104 2 3 1 1 Saving Images As a Series 2 104 2 3 1 2 Saving a Display 2 106 2 3 1 3 Saving Specified Area of Image 2 108 2 3 1 4 Saving Animation Images 2 111 2 3 1 5 File Types Available for Save 2 112 2 3 2 Opening Previously Saved Images 2 119 2 3 3 S...

Page 28: ... Per Channel 2 150 2 4 7 2 Displaying Images of Two Channels Together 2 152 2 4 7 3 Displaying Time Lapse Images 2 153 2 4 7 4 Displaying Multiple Multiple sections Images 2 154 2 4 7 5 Displaying Same Images in Different Display Methods 2 154 2 4 7 6 Re arranging Images Using the Same Display Method 2 156 2 4 7 7 Displaying Different Images Together 2 156 2 4 8 Displaying an Image in Full Screen ...

Page 29: ...e 2 204 2 7 1 1 Display Switching to Built Image 2 204 2 7 1 2 Turning Built Image into Single Image 2 207 2 7 1 3 Turning Built Image into time series image 2 209 2 7 2 Building line images to be viewed in Z direction 2 209 2 8 Viewing 3D Image 2 211 2 8 1 Successive Display of Images 2 212 2 8 1 1 Changing the Successive Display Speed 2 213 2 8 2 Animation 2 214 2 8 3 Building Stereo 3D Images 2...

Page 30: ...r 2 240 2 11 12 Changing the Comment Font 2 240 2 12 Image Output at Printer 2 242 2 13 Merger Extraction of Image Channels 2 244 2 13 1 Setting the Range of Multiple Image Slices 2 244 2 13 2 Merging Image Channels 2 245 2 13 3 Extracting Channels from Image 2 249 2 14 Changing the Chart Display Method 2 253 2 14 1 Chart Panel 2 253 2 14 2 Series Panel 2 261 2 15 Pop up Menus 2 264 Appendix A Lis...

Page 31: ...into a Chart Using EXCEL G 1 Appendix H USER REGISTRATION OF FV500 H 1 Appendix H 1 User Registration H 1 Appendix H 2 Logging into the FV500 H 2 Appendix H 3 Deleting a User H 3 Appendix I Change of Default Folder for File I O PanelI 1 Appendix J List of Functions in the Active Overlays Dialog Box J 1 Appendix J 1 Coordinate Position Data J 1 Appendix J 1 1 X Coordinate J 1 Appendix J 1 2 Y Coord...

Page 32: ...ppendix J 3 Other J 5 Appendix J 3 1 Channel Number J 5 Appendix J 3 2 Objective Power J 5 Appendix J 3 3 Date of Image Capturing J 5 Appendix J 3 4 Time of Image Capturing J 5 Appendix J 3 5 Image File Name J 5 ...

Page 33: ...hting equipment may differ from those shown below For detailed microscope operation procedures refer to the instruction manuals of your microscope Combination with BX51 or BX61 2 Cube display window 1 Light path selector button 3 Analyzer U MDICT3 4 Transmitted light DIC FV5 DICTS WI DICTH with BX61WI optional 5 Filters LBD ND6 ND25 When using BX61WI LBD FFR This figure shows the case of BX 6 Univ...

Page 34: ...on and polarized light observation Rotate the turret to engage the U MDICT3 in the light path for visual transmitted light differential interference observation or transmitted polarized light observation For laser microscope always disengage the U MDICT3 Engaging the U MDICT3 in the light path degrades the image quality 4 Transmitted light DIC FV5 DICTS WI DICTHRA when using BX61WI optional This i...

Page 35: ...ost into the light path Disengaging the FR Frost from the light path may generate interference fringes on an image 6 Universal condenser Condenser for transmitted lighting In addition the rotary turret for the transmitted light DIC prism and the polarizing plate for differential interference observation polarizer are also provided To perform differential interference observation engage the transmi...

Page 36: ...or reflected light observation can be switched with the hand switch for transmitted reflected light 7 Engage the desired cube in the light path for visual fluorescence observation For laser microscopy or visual transmitted light observation press the BF cube switch button of the hand switch 7 This set the cube turret so that no cube is engaged 4 Transmitted light DIC slider FV5 DICT 6 Universal co...

Page 37: ...for laser differential interference observation or visual transmitted light differential interference observation Leaving the FV5 DICT engaged during laser fluorescence observation will degrade resolution somewhat We recommend disengaging the FV5 DICT from the light path when simple laser fluorescence observation is required With transmitted light differential interference observation using an imm...

Page 38: ...on polarizer are also provided To perform differential interference observation engage the transmitted light DIC prism matching the objective in use in the light path For both visual observation and laser differential interference observation To perform visual differential interference observation or laser differential interference observation engage the polarizing plate in the light path 7 Hand s...

Page 39: ...EW Basic Operations 1 7 Page Combination with IX 3 Analyzer FV5 ANI 1 Light path selector 4 Transmitted light DIC slider FV5 DICT optional 2 Cube turret 5 Condenser 6 Filters 7 Intermediate magnification knob Left side view ...

Page 40: ...g plate for use in differential interference observation and polarized light observation Set to the pushed in position to engage the FV5 ANI in the light path for visual transmitted light differential interference observation or transmitted polarized light observation Set to the pulled out position to disengage the FV5 ANI for laser microscopy Engaging the FV5 ANI in the light path degrades image ...

Page 41: ...nsmitted light DIC slider optional matching the objective in use in the light path For both visual observation and laser differential interference observation To perform visual differential interference observation or laser differential interference observation engage the polarizing plate in the light path 6 Filters These filters are used to adjust transmitted light For transmitted observation usi...

Page 42: ...sual observation and side port Set the knob to the position for the desired light path by referring to the following table Light ratios of light paths Knob Position Indication Light Path LSM SP Side port Binocular tube or LSM 100 100 Left side view 1 Light path selector 2 Cube turret 3 Analyzer FV5 ANI 4 Transmitted light DIC slider FV5 DICT optional 6 Filters 5 Condenser ...

Page 43: ... disengage the analyzer from the light path by pulling the lever out High quality image cannot be obtained if the analyzer is left engaged in the light path 4 Transmitted light DIC slider FV5 DICT optional Prism for use in DIC observation For laser DIC observation or visual transmitted light DIC observation engage If fluorescence observation using laser is performed with the FV5 DICT engaged in th...

Page 44: ...arizer for DIC observation are also provided here For DIC observation engage the transmitted light DIC slider optional matching the objective in use in the light path This applies to both visual and laser DIC observations For visual DIC observation or laser DIC observation engage the polarizer in the light path 6 Filter Used in adjustment of the transmitted light illumination ...

Page 45: ...g and execute its function Double clicking To double click place the mouse pointer on the desired function and press the mouse button successively twice To move something Dragging To drag place the mouse pointer on the desired function and while pressing and holding the mouse button move the mouse to the desired destination At the desired destination release the mouse button Dragging by pressing t...

Page 46: ...rge scale Clicking the top or bottom arrow button allows fine adjustment of the value Dragging the square knob allows the value to vary directly Page tab Click to switch the panel for executing the indicated processing function Right clicking a page tab displays the pop up menu of all items under it so the desired one can be selected Buttons Click each button to execute the processing indicated on...

Page 47: ...Control menu box Clicking this box displays a control menu which contains the commands for use in controlling the window Group box The group box groups functions with specific meanings and encloses them in a frame List box Shows the list of available items for selection All items in the list can be displayed by scrolling To select an item in the list double click or drag the item Check box Clickin...

Page 48: ... the laser combiner is used set the optimum ND filters for the laser Section 1 2 7 Select the light path for 100 binocular tube and focus on the specimen Section 1 2 3 Adjust PMT Voltage Sec 2 1 1 4 9 Change the ND filter with a filter with higher transmittance Sec 1 2 7 Set the objective magnification Sec 1 2 8 1 Set the zoom ratio to 1X Sec 1 2 8 2 Set the channels Sec 1 2 8 3 Set the highest sc...

Page 49: ... 2 7 Set the objective magnification Sec 1 2 8 1 Set the zoom ratio to 1X Sec 1 2 8 2 Set the channels Sec 1 2 8 3 Set the highest scan speed Sec 1 2 8 4 Select the XY observation mode Sec 1 2 8 5 Perform repeated scanning Sec 1 2 8 6 If an image is displayed Acquire an image Sec 1 2 9 Save the image Sec 1 2 10 Exit from the FLUOVIEW software Sec 1 2 11 Turn the system power OFF Sec 1 2 12 Image i...

Page 50: ...turned ON in interlocking with the power of the computer Power supply unit Monitor Refer to the instruction manual of your monitor Since these units are permanently connected to the computer leave their power switches set to the ON position It takes about 2 minutes after power supply unit turns ON until it is initialized Before proceeding to any other operation wait for about 2 minutes after turni...

Page 51: ...d should exceed 10 minutes when the laser system in use uses an Ar laser or exceed 30 minutes when it uses a Kr laser or Green HeNe laser 2 4 UV Ar laser When using the system for the first time check that the remote cable is connected the POWER switch on the front panel of the chiller is set to O OFF its COOL DOWN CYCLE switch is set to I ON and the remote operation mode is set Fig 1 3 Chiller Fr...

Page 52: ...e to the ON position 4 Turn the ON button of the remote interface module ON After the key is set to ON it takes tens of seconds before the laser oscillation starts To stabilize the laser beam output we recommend leaving the system for warm up after turning the laser power ON The warm up period should exceed 10 minutes when the laser system in use uses an Ar laser or exceed 30 minutes when it uses ...

Page 53: ... the transmitted light detector is used Turning the monitor ON Refer to the instruction manual of your monitor As this unit should be connected to the power outlet unit leave its power switch in the ON position Turning the microscope ON Refer to the instruction manual of the microscope combined with the system As this unit should be connected to the power outlet unit leave its power switch in the ...

Page 54: ...he following wait for more than 2 minutes after turning the microscope and power supply unit ON When using the AX70A and U MCB start the software while the MAIN screen of U MCB is displayed 1 Input the user name and password and log onto Windows NT Input the user name authorized as an Administrator 2 Double click the FLUOVIEW icon on the desktop When more than one user uses the FV500 each user sha...

Page 55: ...Getting Started FLUOVIEW Outline of LSM Observation Procedures 1 23 Page 3 The following window appears when the FLUOVIEW software starts Fig 1 3 Window at Start up ...

Page 56: ...ub panel 2 Select the BI button in the Light Path group box The BI button looks pushed in to indicate that it is selected 3 Push the cube button 3 of the hand switch 2 to engage the optimum cube for specimen dye In the cube display window 1 the selected cube is displayed 4 Focus on the specimen by looking into the eyepiece Be sure to adjust the diopter of the eyepiece in advance Refer to the instr...

Page 57: ...heck box is checked for this may damage the Z Motor The specimen may float during oil immersed observation In this case prepare an optional clip for immersion objective and attach it as shown on the left If you want to use a differential interference unit in transmitted light observation refer to the instruction manual of your microscope With transmitted light differential interference observation...

Page 58: ...ion button 2 on the hand switch for transmitted reflected light 1 4 Focus on the specimen by looking into the eyepiece Be sure to adjust the diopter of the eyepiece in advance Refer to the instruction manual of the AX microscope When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume then focus on th...

Page 59: ... immersed observation In this case prepare an optional clip for immersion objective and attach it as shown on the left To observe a TV image when the system is combined with a BX or AX microscope click the TV button in the Light Path group box in the Optics sub panel of the Acquire panel TV button NOTE NOTE ...

Page 60: ...e instruction manual of the IX50 70 microscope When the Z motor is in use clear the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume then focus on the specimen by operating the fine focus adjustment knob of the microscope Be sure not to operate the fine focus adjustment knob while the Locked check box is checked for this may dama...

Page 61: ...e right side of microscope to LSM 2 From the index tabs on the bottom right of the Acquire panel select the Optics sub panel Fig 1 4 Optics Sub panel 3 Click the BI button in the Light Path group box The BI button looks pushed in to indicate that it is selected 4 Rotate the cube turret 2 to engage the cube matching the specimen dye in the light path 1 ...

Page 62: ...ar the check mark in the Locked check box in the Z Stage sub panel in the Acquire panel see section 2 2 1 4 7 of this volume before bringing the specimen into focus using the focus adjustment knobs of the microscope If the fine focus adjustment knob of the microscope is rotated while the Locked check box is checked the Z motor may be destroyed The specimen may float during immersed observation In ...

Page 63: ... box The LSM button looks pushed in to indicate that it is selected When scanning is started while the BI button is selected the LSM light path is selected automatically It is switched back to the visual observation automatically when scanning completes 3 Push the hand switch button to 8 set to be displayed in the cube display window 1 on the reflected light fluorescence vertical illuminator 4 Whe...

Page 64: ...r the objective in the light path by operating the universal condenser 5 6 For transmitted light observation disengage any filter 4 from the light path When you perform transmitted observation using laser with BX61WI use the filter knob to disengage the LBD from the light path and engage the FR Frost into the light path Disengaging the FR Frost from the light path may generate interference fringes...

Page 65: ... FV5 ANU analyzer 3 is in use disengage it from the light path by setting the switch to the pulled out position 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 4 by setting the switch to the pulled out position When transmitted light differential interference observation or simultaneous fluorescence transmitted light differential interference ob...

Page 66: ...ting started FLUOVIEW Outline of LSM Observation Procedures 1 34 Page 5 Filters LBD ND6 ND25 6 Universal condenser 4 Transmitted light DIC slider FV5 DICT optional 3 Analyzer FV5 ANU optional 2 Cube turret ...

Page 67: ...vertical illuminator mirror cube housing 1 by pressing the desired cube conversion button 6 on the U SHTR hand switch 4 When the FV5 ANA analyzer 2 is in use disengage it from the light path by setting the switch to the pulled out position 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 3 by setting the switch to the pulled out position When tra...

Page 68: ...servation Procedures 1 36 Page 6 1 Universal vertical illuminator mirror cube housing AX URBC 2 Analyzer FV5 ANA 3 Transmitted light DIC slider FV5 DICT 4 Filters Filter lever ND Filter levers IF550 LBD 5 Universal condenser Hand switch U HSTR ...

Page 69: ... the visual observation automatically when scanning completes 3 When the FV5 ANA analyzer 1 is in use disengage it from the light path by setting the switch to the pulled out position 4 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider from the light path by pulling it out When the fluorescence transmitted light simultaneous interference observation...

Page 70: ...Getting started FLUOVIEW Outline of LSM Observation Procedures 1 38 Page 1 Analyzer FV5 ANA 2 Transmitted DIC slider FV5 DICT ...

Page 71: ...ted DIC slider 4 by setting the switch to the pulled out position When transmitted light differential interference observation or simultaneous fluorescence transmitted light differential interference observation is required engage the FV5 DICT and the optimum transmitted light DIC slider for the objective in the light path by operating the universal condenser 5 With simultaneous fluorescence trans...

Page 72: ...Getting started FLUOVIEW Outline of LSM Observation Procedures 1 40 Page 1 Light path selector 2 Cube turret 3 Analyzer FV5 ANI 4 Transmitted DIC slider FV5 DICT 5 Condenser 6 Filters Left side view ...

Page 73: ...ertical fluorescence illuminator to 4 If the FV5 ANI analyzer 3 is engaged in the light path pull the lever out to disengage it 5 When only fluorescence observation is required disengage the FV5 DICT transmitted light DIC slider 4 from the light path When performing transmitted DIC observation or fluorescence transmitted light DIC simultaneous observation engage the FV5 DICT as well as the transmi...

Page 74: ...started FLUOVIEW Outline of LSM Observation Procedures 1 42 Page Left side view 1 Light path selector knob 2 Cube turret 3 Analyzer FV5 ANI 4 Transmitted light DIC slider FV5 DICT optional 6 Filter 5 Condenser ...

Page 75: ...ire panel Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method Clear button Clear the set dyeing method Prev button Sets the dyeing method which was set last time by clicking the Apply button Apply button Applies the dyeing method dragged in the Selected Dyes group box to the Ch group in the Acquire...

Page 76: ...or acquiring fluorescence is set automatically according to the switched filter And the dyeing method is shown in the Ch group box The Confocal Aperture value is also set automatically according to the wavelength and the objective for every channel If you have changed the objective click the Apply button in the Dyes sub panel The Conforcal Aperture value is set appropriately TIP TIP ...

Page 77: ...s manually check box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually ch...

Page 78: ...window The following table shows the possible combinations of the barrier and excitation filters Laser Combination Excitation Filter Spectral Filter 1 Spectral Filter 2 Spectral Filter 3 Barrier Filter 1 Barrier Filter 2 Barrier Filter 3 Barrier Filter 4 Ar 488 DM351 488 Fix 1 Mirror 2 Glass 3 SDM560 4 5 1 BA505 2 BA505 550 3 4 5 6 7 1 BA560 2 BA610 3 4 5 6 7 Ar HeNe G 488 543 DM488 543 Fix9 1 Mir...

Page 79: ...505 550 3 4 5 6 7 1 BA585 2 BA610 3 BA585 615 4 5 6 7 1 BA660 2 3 4 5 6 7 UV Ar HeNe G HeN e R 351 488 543 633 1 BS2 8 2 DM351 488 3 DM351 543 4 DM351 5 DM488 543 6 DM488 543 633 7 1 Mirror 2 Glass 3 SDM490 4 5 1 Mirror 2 Glass 3 SDM560 4 5 1 Mirror 2 Glass 3 SDM630 4 5 1 BA400 470 2 3 4 5 6 7 1 BA505 2 BA505 525 3 4 5 6 7 1 BA565 2 BA610 3 BA560 600 4 5 6 7 1 BA660 2 3 4 5 6 7 UV Ar Kr HeNe R 351...

Page 80: ...e ND filter scale of each laser according to the levels of specimen brightness fluorescence crosstalk and fading Fig 1 11 Lasers Sub panel When the Green HeNe laser is used try ND of 50 by setting the Intensity scale in the Lasers group box to 50 With other lasers try ND of 10 first Laser Intensity group box Set the ND of the lasers in the scales The number of displayed lasers varies depending on ...

Page 81: ... list on the center of the Acquire panel select the objective being used with the microscope With a combination using a microscope other than the AX70A the measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use If you change the objective click the Apply button in the Dyes sub panel The Conforcal Aperture value...

Page 82: ...annels are ready for image acquisition If the check box of a channel is not check marked check it to make the channel ready To display the information on all channels simultaneously right click the boundary between channel display boxes Click the boundary again to return to the original display 1 2 8 4 Setting the Highest Scan Speed 1 Set the scan speed to the fastest speed by using the scale in t...

Page 83: ...ne skipped scan As a result the acquired images become coarse 1 2 8 5 Setting the XY Observation Mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Scan Mode group box in the Acquire panel select 800 by 600 from the Size drop down list 3 In the Acquire panel select the XY observation mode option button NOTE TIP Focus Mode group box X2 option button Acq...

Page 84: ... coarse Do not move FLOUVIEW FV500 Menu while acquiring an image 1 2 8 7 Setting the Cross section to be Observed While acquiring image move the Z stage to select the cross section to be observed 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel Fig 1 12 Z Stage Sub panel XY Repeat button Focus button Current Pos text box Shows the current posit...

Page 85: ...ation targets are concentrated in a narrow area or when observation of a specific area detail is required the image of a limited area can be selected Fig 1 13 Pan Zoom Group Box The 4 buttons represent directions and clicking a button moves the acquired image area in the direction indicated by the button Clicking the square button on the center returns the acquired image area to the center Clickin...

Page 86: ...the time required for image acquisition When the scan speed is decreased during fluorescence observation the saturation of fluorescence may darken the image of certain types of specimens In this case increase the scan speed and increase the PMT Voltage or use accumulation in scanning 1 2 8 10 Stopping Repeated Scanning After the brightness and gain have been adjusted select the STOP SCAN button in...

Page 87: ...SM Observation Procedures 1 55 Page 1 2 9 Acquiring Image Select the Once button The acquired image will be displayed in the Live panel Fig 1 14 Image Acquired in the Live Panel 1 2 10 Saving Image 1 Display the File I O panel Once button ...

Page 88: ...ttons to select the images to be saved The selected images will be saved under the conditions set for each channel Example When only the image of Channel 1 is displayed only the image of Channel 1 will be saved 3 Click Experiment button in the Save group box The Save Experiment As dialog box will open Fig 1 15 Save Experiment As Dialog Box 4 Enter the file name in the File name text box 5 Select F...

Page 89: ...t the Close all experiments and log in as a different user option button and click the Yes button The dialog box asking whether to save the observation data or not appears If you want to save the data click the Yes button or the No button if saving is unnecessary It returns to the FLUOVIEW login screen 4 To shut down Windows select the Windows Start button to display the Start menu and select the ...

Page 90: ...er unit HeNe Green Red laser Turn the key to the OFF position UV Ar laser 1 1 Turn the key switch on the UV laser power supply to the O OFF position 1 2 Set the MAIN POWER lever on the UV laser power supply to the O OFF Chiller is switched to auto cooling operation by interlocking When it is left in auto cooling it will automatically turn off in about 15 minutes While the heat exchanger is running...

Page 91: ... OFF Wait till LASER OFF message appears on digital display For about 5 minutes to cool it down 2 Turn the key switch of remote interface module to the OFF position 3 Turn the key switch of laser controller to the OFF position 4 Turn the rocker switch of laser controller to 0 OFF Turning the laser power off by force may damage the laser tube Never omit the step 1 the process for cooling down excep...

Page 92: ...thod for displaying and consulting the online help on the functions and operation procedures The figure below shows the initial display table of contents of the FLUOVIEW Online Help window To display this window click the Help button in the toolbar at the bottom left of the screen Some words in the displayed information are shown in enhanced display underscored or colored green Clicking one of the...

Page 93: ...yes list box in the Selected Dyes group box to the field immediately above the list box 3 Click the Apply button to apply the selected dyeing method to the Ch group box on the upper part of the Acquire panel TIP Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method Clear button Clear the set dyeing m...

Page 94: ...Dyes list box and the Apply button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is shown in the Ch group box The Confocal Aperture value is also set automatically according to the wavelength and the objective per channels TIP ...

Page 95: ...eck box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually check box ...

Page 96: ...ed FLUOVIEW Online Help 1 64 Page 1 3 2 1 Configuring the Microscope 1 Display the Acquire panel Scan Z Stage Time Series Optics Lasers sub panels These are used to set the information required for image acquisition ...

Page 97: ...ig 1 19 Microscope Configuration Window Scope Control button Displays useful information for the system setup Analyzer box Represents the FV5 ANA analyzer optional Turret Cube box Represents the cube turret Observation group box LSM represents the laser scanning microscopy light path and BI represents the visual observation light path Fluorescence represents fluorescence observation for both LSM a...

Page 98: ...ation window is designed to give guidance on system configuration The red blinking light does not stop even after the indicated configuration point has been changed 5 While looking into the microscope move the stage and check the observed image 1 3 2 2 Configuring the Microscope Combination with BX51 BX61 When the combination with BX51 or BX61 is in use the microscope and scan unit FV500 can be co...

Page 99: ... path Condenser Turret group box Clicking the universal condenser automatically switches the turret Aperture Iris group box Changes the AS value EPI lamp Indicates the EPI lamp BX Options group box Used for optional BX settings Link Setting button Selects the function to change settings corresponding to the change of BX settings Load Setting button Loads the BX settings already registered to refle...

Page 100: ...etting file which you want to read Click the OK button to close the dialog box Checking here links the objective in the Nosepiece group box with the condenser turret Checking here escapes the stage when the objective is selected and changed in the Nosepiece group box Checking here disengages the top lens from the light path when the objective of 4 or lower magnification is selected in the Nosepiec...

Page 101: ...ng the Save Setting button displays the Save As dialog box as shown below To change the save destination drive or directory use the Save in drop down list Enter the setting file name into the File name text box Select the Save button to close the dialog box ...

Page 102: ...tion with the AX70A the microscope and scan unit can be configured from the FLUOVIEW software Use the following procedure for this 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Fig 1 20 Optics Sub panel Scope Control button Click to start the system setup ...

Page 103: ...CB After the setup using the U MCB click this button again to apply the set up contents in the AX Control window Cube group box Select the excitation filter from the drop down list The selection from the drop down list is possible when the Reflected Fluorescence Transmitted Differential Interference or Reflected Fluorescence button is selected in the Observation mode group box Shutter group box Op...

Page 104: ... Lamp group box The transmitted light lamp is set to OFF FILTER group box The value of ND is set to 100 FILTER group box Both color correction filters are set to OUT 1 3 2 4 Configuring the Filters The barrier filters excitation filter and beam splitter are set automatically to the light path according to the dyeing method selected for the specimen Use the following procedure to change these filte...

Page 105: ...tion we recommend you making Stby mode The laser oscillation stops when the time shown in this box has elapsed after the end of laser scanning The laser oscillation stops when the time shown in this box has elapsed after the end of laser scanning The laser is suspended as standby mode using Ar or Kr laser or the laser oscillation stops using UV Ar laser When the time shown in this box has elapsed ...

Page 106: ...according to the selected dyeing method Use the following procedure to change the pinhole diameters 1 Display the Acquire panel Ch group box Sets whether the image of each channel is to be acquired or not the PMT voltage Gain and Offset Right clicking the mouse inside this group box displays the Ch group boxes of all channels ...

Page 107: ...d Offset LED sliders Set these values independently Increasing the PMT Voltage improves the sensitivity Increasing the Gain brightens the image Increasing the Offset darkens the image Ch1 Ch2 Ch3 Ch4 and Ch5 group boxes Select the channels from which you want to acquire images button Click to display the pinhole diameter and the laser ND filter setting ...

Page 108: ...ndently Increasing the PMT Voltage improves the sensitivity Increasing the Gain brightens the image Increasing the Offset darkens the image Ch1 Ch2 Ch3 Ch4 and Ch5 group boxes Select the channels from which you want to acquire images button Click to close each group box showing the pinhole diameter and laser ND filter value The pinhole diameter and laser ND filter value are valid even before the g...

Page 109: ...ider increases or decreases the laser ND value by 1 Each click of the slider section of the Laser ND LED slider varies the laser ND value by 5 To set to the Confocal Aperture value to its primal value click the Apply button in the Dyes sub panel of the Acquire panel Clicking this button allows fine adjustment of the value Clicking this field allows the value to be changed on a large scale The ND v...

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Page 111: ...VIEW system by acquiring an image or opening an image from a file The procedures for the subsequent operations such as image processing are not in question here For the detailed operation method of each item in the procedure see the section specified in parentheses The following is the general operation procedure of FLUOVIEW Many other functions that are not shown in the following are also availab...

Page 112: ...lay Section 2 8 2 Continuous display Frame by frame display Section 2 8 1 z Observation of image change over time Side by side image display Section 2 9 1 Continuous display Frame by frame display Section 2 9 2 Measure the image z Measurement of image shape Length measurement Section 2 6 3 1 Area measurement Section 2 6 3 2 z Measurement of image intensity value and distribution Intensity on a lin...

Page 113: ...he following observation modes Configure the system and confirm the images to be observed The system configuration and image confirmation are described in details in section 1 2 Outline of LSM Observation Procedures in the OPERATION Volume Refer to this section as required TIME With this system the observation by XY scanning is called simply as XY observation A B TIME TIME Save the acquired data i...

Page 114: ...ge Acquisition in Other Observation Modes as well as the following procedure Set the acquisition parameters Section 2 2 1 4 z Observation mode z Scanning speed z Image brightness Acquire an image in the XY observation mode Section 2 2 1 5 XY observation Section 2 2 1 Perform accumulation Section 2 2 1 6 After Configure the system and confirm the images to be observed Go to Save the acquired data i...

Page 115: ...uired image in a file measure the intensity distribution and edit data with Excel Specify the area to be measured Measure the intensity distribution Section 2 6 2 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 1 2 12 Save the measurement data in the Excel format Section 2 10 1 Turn power ON and start the FLUOVIEW software Sections 1 2 1 1 2 2 Open an image in a file Section 2 3...

Page 116: ...IEW software and turn power OFF Sections 1 2 11 1 2 12 Output the image at the printer Section 2 12 Open an image in a file Section 2 3 2 Exit from the FLUOVIEW software and turn power OFF Sections 1 2 11 1 2 12 Open an image in a file Section 2 3 2 Turn power ON and start the FLUOVIEW software Sections 1 2 1 1 2 2 Convert the contrast Section 2 5 2 Draw comment text on the image Section 2 11 Disp...

Page 117: ...tion 2 7 Page 2 2 Image Acquisition Confirm the image to be acquired using the microscope and acquire its image using FLUOVIEW The image can be saved in a file as required Do not move FLOUVIEW FV500 Menu while acquiring an image NOTE ...

Page 118: ...Set the channel to be acquired Section 2 2 1 4 3 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION Set the observation range Section 2 2 1 4 8 in OPERATION Adjust the Z position to observe the desired multiple sections Section 2 2 1 4 7 in OPERATION Set a lower s...

Page 119: ... one of the displayed observation modes Scan Speed group box Sets the scan speed Sliding to the left of the scale increases the scan speed and to the right decreases it PMT Gain and Offset LED sliders These can be adjusted independently Increasing the PMT voltage improves the sensitivity Increasing the Gain brightens the image Increasing the Offset darkens the image Ch1 Ch2 Ch3 Ch4 and Ch5 check b...

Page 120: ...ct the desired items from this list and drag them to the field above it to select the dyeing method Clear button Clear the set dyeing method Prev button Sets the dyeing method which was set last time by clicking the Apply button Apply button Applies the dyeing method dragged in the Selected Dyes group box to the Ch group in the Acquire panel Place the pointer on the icon displayed in the Selected ...

Page 121: ... list box and the Apply button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is shown in the Ch group box The Confocal Aperture value is also set automatically according to the wavelength and the objective per channels If you change the objective click the Apply button in the Dyes sub panel The Conforcal Aperture value is...

Page 122: ...eck box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually check box ...

Page 123: ...quire panel select the Optics sub panel Fig 2 4 Optics Sub panel 4 Select the Scope Control button at the bottom of the panel The window as shown below will appear in case of a combination with the IX Fig 2 5 Microscope Configuration Window Scope Control button Displays useful information for the system setup ...

Page 124: ... system configuration setup of cube turret etc by following the guidance given by the red blinking light For the operation of the lever see section 1 1 1 Microscope in this volume The Microscope Configuration window is designed to give guidance on system configuration The red blinking light does not stop even after the indicated configuration point has been changed 7 While looking into the microsc...

Page 125: ...th according to the dyeing method selected for the specimen To change filters use the following procedure 1 From the panel page tabs shown on the bottom right of the Acquire panel select the Optics sub panel Fig 2 6 Optics Sub panel SU Control button Click to display the filter and laser types to be set Combination with the laser combiner sets up the laser automatically ...

Page 126: ...r each channel Laser Unit group box Shows the type of laser to be used With the laser combiner operation the laser type is set and displayed automatically When the On button is pressed in the laser is oscillating the beam When the Stby button is pressed in the laser is not oscillating When the Auto standby check box is checked the After text box appears below it When not using laser for a long tim...

Page 127: ...asers sub panel set the ND filter scale of each laser according to the levels of specimen brightness fluorescence crosstalk and fading When the HeNe Green laser is used try ND of 50 by setting the Intensity scale in the Lasers group box to 50 With other lasers try ND of 10 first Clicking this field allows the ND value to be changed on a large scale The ND values which are usually used are displaye...

Page 128: ...pe other than the AX70A the measurement results will be inaccurate if the objective magnification set here does not match the actual magnification of the objective in use If you change the objective click the Apply button in the Dyes sub panel The Conforcal Aperture value is set appropriately 2 Setting the Zoom Ratio to 1X 1 Use the Zoom scale in the Acquire panel to set the zoom ratio to X1 Fig 2...

Page 129: ... the image acquisition 3 In the Channel 3 group box check the check box showing the applicable dyeing method to ready the image acquisition 4 In the Channel 4 group box check the check box showing the applicable dyeing method to ready the image acquisition To display the information on all channels simultaneously right click the boundary between channel display boxes Click the boundary again to re...

Page 130: ...option button in the Focus Mode group box The focus mode is enabled when acquiring images using the Focus button The Focus function reduces the scanning time by line skipped scan As a result the acquired images become coarse Scan Speed group box Set the scan speed by clicking a point on the scale line TIP Focus Mode group box X2 option button Acquires image at twice the highest speed X4 option but...

Page 131: ...button The Focus function reduces the scanning time by line skipped scan As a result the acquired images become coarse 7 Setting the Multiple sections to be Observed While acquiring image move the Z stage to select the multiple sections to be observed From the panel page tabs shown on the bottom right of the Acquire panel select the Z Stage sub panel Fig 2 9 Z Stage Sub panel XY Repeat button Focu...

Page 132: ... Configuration for detailed operations 1 Check the Locked check box in the Z Stage sub panel Do not turn the fine focus adjustment knob while the Locked check box is checked for this may damage the Z motor 2 While observing the image in the Live panel locate the plane to be observed by displacing the stage using the Z stage coarse adjustment and Z stage fine adjustment buttons in the Z Stage sub p...

Page 133: ...the left of the Zoom scale represents the field visible through the microscope and the blue frame indicates the acquired image area Move the blue frame inside the circle using the Pan buttons so that the desired observation targets are displayed in the Live panel The 4 buttons represent directions and clicking a button moves the acquired image area in the direction indicated by the button Clicking...

Page 134: ...his value can be set independently from the Offset For example When the acquired image is dark or the observation targets are hardly visible increase PMT voltage to improve the sensitivity If the image resulting from this becomes too bright first try decreasing PMT voltage slightly If the image background is still too bright increase Offset to darken the background As this also reduces the brightn...

Page 135: ...by 10V Each click of the or button of the Gain LED slider increases or decreases the Gain by X0 5 Each click of the slider section of the Gain LED slider varies the Gain by X0 5 Each click of the or button of the Offset LED slider increases or decreases Offset by 1 Each click of the slider section of the Offset LED slider varies the Offset by 5 When the light incident to photomultiplier tube is to...

Page 136: ...d during fluorescence observation the saturation of fluorescence may darken the image of certain types of specimens In this case increase the scan speed and increase the PMT Voltage or use accumulation in scanning 11 Stopping Repeated Scanning 1 After the brightness and gain have been adjusted select the STOP SCAN button in the Acquire panel to stop scanning temporarily 2 2 1 5 Acquiring Image 1 S...

Page 137: ...quisition when any intensity value on the image reaches the peak 4095 This operation is effective for acquiring an image with the dark lower part in the XYZ observation and observation of an extremely dark image Kalman Accumulation Algorithm Every time an image is acquired the pixel values are rewritten based on the following formulae where it is assumed that n number of image acquisitions l n Res...

Page 138: ...ak option button 3 When Kalman is selected enter the accumulation count in the text box And select the Frame Kalman option button displayed The accumulation count can be set up a maximum of 63 times When 0 is set as the number of times of accumulation an ordinary image acquisition is to be performed Filter group box Select the accumulation mode Two accumulation modes Kalman and Accumulate To Peak ...

Page 139: ...uiring Image in Accumulation Mode Line mode Image acquisition in the line mode can be performed when you use the FV300 system with AOTF FV5 COMBA 1 From the page tabs on the bottom right of the Acquire panel select the Scan sub panel Fig 2 12 Scan sub panel 2 In the Filter group box select the Kalman option button NOTE Filter group box Select the accumulation mode Two accumulation modes Kalman and...

Page 140: ... frame mode an image is created by accumulation after scanning is performed per image at specified times Meanwhile in the line mode scanning is performed per line at specified times and after accumulation is performed the next line is to be scanned Each mode can be selected according to its ability the frame mode is adequate for observing fixed sells and the line mode is adequate for observing liv...

Page 141: ...ection will be focused on the image acquisition operations in the XZ observation mode that are not used in the XY observation modes which are the operations enclosed in in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections ...

Page 142: ...eed Section 2 2 1 4 10 in OPERATION Stop repeated scanning Section 2 2 1 4 11 in OPERATION Set the observation mode Section 2 2 2 1 2 in OPERATION Acquire image Section 2 2 2 1 5 in OPERATION Set the observation line Section 2 2 2 1 3 in OPERATION Set the range of the multiple sections to be observed the Z direction scanning range Section 2 2 2 1 1 in OPERATION Set the numbers of Z direction steps...

Page 143: ... Stop Z and Start Z values Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Set button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning ran...

Page 144: ...locate the bottom edge of the range to be observed by moving up the stage using the Z stage coarse adjustment and Z stage fine adjustment buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the Z stage coarse adjustment and Z stage fine adjustment buttons 5 When the ...

Page 145: ... scale of the acquired image is identical to the horizontal scale The number of steps calculated by the system may be erroneous unless the XZ observation mode has been set previously The number of acquired images shown in the Slices text box can also be input from the keyboard After setting Start Z Z direction scan start position and Stop Z Z direction scanning stop position input the desired numb...

Page 146: ...t the channel to be acquired Section 2 2 1 4 3 in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION Set the observation range Section 2 2 1 4 8 in OPERATION Adjust the Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Set a lower scan sp...

Page 147: ... times successively at the set scanning speed For reference the time required for the image acquisitions is shown in the frame on the bottom left of the Scan Speed group box The number of image acquisitions 2000 times can be changed by changing the value in the N text box in the Time Series sub panel While acquiring an image in the XT observation mode clicking the STOP SCAN button changes the butt...

Page 148: ...the image acquisition operations in the XZT observation mode that are not used in the XY observation modes which are the operations enclosed in in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections ...

Page 149: ... OPERATION Set the observation line Section 2 2 2 3 3 in OPERATION Set the numbers of Z direction steps and acquired image slices Section 2 2 2 3 4 in OPERATION Adjust the Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Adjust the image brightness Section 2 2 1 4 9 in OPERATION Set the zoom ratio to 1X Section 2 2 1 4 2 in OPERATION Set the interval time Section 2 2 ...

Page 150: ... the observed cross section Z direction scanning range Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Set button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning range Set button Sets the current stage position as the scanning stop position of the rang...

Page 151: ... text box will show the scan start position of the range of the multiple sections to be observed Z direction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be observed by moving up the stage using the Z stage coarse adjustment and Z stage fine adjustment buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope ...

Page 152: ...button in the Step Size text box The number of steps shown in the Step Size text box has been calculated by the system so that the depth scale of the acquired image is identical to the horizontal scale The number of steps calculated by the system may be erroneous unless the XZT observation mode has been set previously The number of acquired images shown in the Slices text box can also be input fro...

Page 153: ...e Interval text box shows Free Run message Using button also sets the Interval text box to Free Run 6 Setting the number of scans 1 Set the number of scans using the or button in the N text box in the Time Series sub panel Rest Time text box Shows the time after end of acquisition of an image until the start of next acquisition N text box Set the number of scans using the or button or by input fro...

Page 154: ...ge in the XZT observation mode the buttons at the upper part of the Acquire panel changes as shown below Click the Append Next button to append an image An image will be acquired with the same number of steps as the image acquired immediately before and appended to it Click the Series Done button when it is not required to append an image Append Next button Acquires another image and appends it to...

Page 155: ...the image acquisition operations in the XYZ observation mode that are not used in the XY observation modes which are the operations enclosed in in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections ...

Page 156: ...in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Re adjust the image brightness Section 2 2 1 4 9 in OPERATION Stop repeated scanning Section 2 2 1 4 11 in OPERATION Set the observation mode Section 2 2 2 4 3 in OPERATION Acquire image Section 2 2 2 4 4 in OPERATION Set the range of the cross section to be observed the Z direction scanning range Section 2 2 2 4 1 in OPERATION Se...

Page 157: ...p Z and Start Z values Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Set button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning range S...

Page 158: ...Start Z text box will show the scan start position of the range of the multiple sections to be observed Z direction scanning range 4 While observing the image in the Live panel locate the bottom edge of the range to be observed by moving up the stage using the Z stage coarse adjustment and Z stage fine adjustment buttons in the Z Stage sub panel When using the FLUOVIEW system with an inverted micr...

Page 159: ...f steps 3 Setting the observation mode 1 In the Scan Mode group box in the Acquire panel select the Surface option button 2 In the Acquire panel select the XYZ observation mode option button 4 Acquiring image 1 Click the XYZ button in the Acquire panel The acquired image will be displayed in the Live panel While acquiring an image in the XYZ observation mode clicking the STOP SCAN button changes t...

Page 160: ...changes as shown below Click the Append Next button to append an image An image will be acquired with the same number of steps as the image acquired immediately before and appended to it Click the Series Done button when it is not required to append an image Append Next button Acquires another image and appends it to the image acquired immediately before Series Done button Determines the acquired ...

Page 161: ... in OPERATION Set the highest scan speed Section 2 2 1 4 4 in OPERATION Perform repeated scanning Section 2 2 1 4 6 in OPERATION Set the XY observation mode Section 2 2 1 4 5 in OPERATION Set the observation range Section 2 2 1 4 8 in OPERATION Adjust the Z position to observe the desired cross section Section 2 2 1 4 7 in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Stop repea...

Page 162: ...l 2 Set the interval time using the or button in the Interval text box An image with no interval can be acquired by entering 0 in the Interval text box In this case the Interval text box shows Free Run message Using button also sets the Interval text box to Free Run Rest Time text box Shows the time after end of acquisition of an image until the start of next acquisition N text box Set the number ...

Page 163: ...ing the focusing to be deviated To acquire precise image data it is recommended to set the power switch of the transmitted light illumination to O OFF While acquiring an image in the XYT observation mode clicking the STOP SCAN button changes the buttons at the upper part of the Acquire panel as shown below The Resume button restarts image acquisition at the frame next to the frame where the acquis...

Page 164: ...the image acquisition operations in the XYZT observation mode that are not used in the XY observation modes which are the operations enclosed in in the chart on the next page For other operations see section 2 2 1 Image Acquisition in XY Observation Mode The details of each operation will be described in the subsequent sections ...

Page 165: ...8 in OPERATION Set a lower scan speed Section 2 2 1 4 10 in OPERATION Re adjust the image brightness Section 2 2 1 4 9 in OPERATION Stop repeated scanning Section 2 2 1 4 11 in OPERATION Set the observation mode Section 2 2 2 6 3 in OPERATION Acquire image Section 2 2 2 6 6 in OPERATION Set the numbers of Z direction steps and acquired image slices Section 2 2 2 6 2 in OPERATION Adjust the Z posit...

Page 166: ...p Z and Start Z values Stop Z text box Shows the scan stop position in the range of the observed cross section Z direction scanning range Start Z text box Shows the scan start position in the range of the observed cross section Z direction scanning range Set button Sets the current stage position as the scanning start position of the range of the observed cross section Z direction scanning range S...

Page 167: ... in the Z Stage sub panel When using the FLUOVIEW system with an inverted microscope locate the upper edge of the range to be observed by moving up the revolving nosepiece using the Z stage coarse adjustment and Z stage fine adjustment buttons 5 When the position is located click the Set button The Stop Z text box will show the scanning stop position of the range of the multiple sections to be obs...

Page 168: ... the or button in the Interval text box An image with no interval can be acquired by entering 0 in the Interval text box In this case the Interval text box shows Free Run message Using button also sets the Interval text box to Free Run Fig 2 19 Time Series Sub panel N text box Set the number of scans using the or button or by input from the keyboard Interval text box Set the interval time using th...

Page 169: ...d by heat causing the focusing to be deviated To acquire precise image data it is recommended to turn off the power of the transmitted light While acquiring an image in the XYZT observation mode clicking the STOP SCAN button changes the buttons at the upper part of the Acquire panel as shown below The Resume button restarts image acquisition at the frame next to the frame where the acquisition is ...

Page 170: ...group box on the upper part of the Acquire panel When the dyeing method is selected from the Available Dyes list box and the Apply button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is shown in the Ch group box Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the ...

Page 171: ...eck box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually check box ...

Page 172: ...ge acquisition In the Acquire panel make sure that the check box in the Ch1 Ch2 Ch3 Ch4 group box that shows the dyeing method is check marked to indicate that the corresponding channel is ready for image acquisition z Set the image brightness Adjust the brightness of the image of the image acquisition channel by using the PMT Offset and Gain LED sliders in the Acquire panel For details see to sec...

Page 173: ...e Acquire panel When the dyeing method is selected from the Available Dyes list box and the Apply button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is shown in the Ch group box Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyei...

Page 174: ...eck box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually check box ...

Page 175: ...the Acquire panel make sure that the check box in the Ch1 Ch2 Ch3 Ch4 group boxes that show the dyeing methods are check marked to indicate that the corresponding channels are ready for image acquisition z When observing a fluorescence image s simultaneously also set the brightness of the fluorescence image s Adjust the brightness of the image of each channel by using the PMT Offset and Gain LED s...

Page 176: ...thod is selected from the Available Dyes list box and the Apply button is clicked a channel for acquiring fluorescence is set automatically according to the changed filter And the dyeing method is shown in the Ch group box Available Dyes list box Lists the available dyes Select the desired items from this list and drag them to the field above it to select the dyeing method Clear button Clear the s...

Page 177: ...eck box in the Dyes sub panel 2 Select the dyeing method in the Available Dyes list box and drag it directly to the field of the Ch check box 3 After dragging the icon appears on the right of the Ch check box and the dyeing method is set Dragging the icon to the out of the Ch check box field cancels the setting of the dyeing method Icon The dyeing method is set Assign dyes manually check box ...

Page 178: ...bulb of the microscope 1 From the page tabs on the bottom right of the Acquire panel select the Optics sub panel Fig 2 23 Optics Sub panel 2 Click the Trans Lamp button to turn the lamp off set the button to the non pressed status z In the Acquire panel make sure that the Transmitted check box in the Ch5 group box is check marked to indicate that Ch3 is ready for image acquisition Trans Lamp butto...

Page 179: ... PMT Offset and Gain LED sliders in Ch5 group box to adjust the brightness of the transmitted image For details see section 2 2 1 4 9 Adjusting the Image Brightness in the OPERATION volume z When observing a fluorescence image s simultaneously also set the brightness of the fluorescence image s Adjust the brightness of the image of the transmitted image acquisition channel s by using the PMT Offse...

Page 180: ... The frame can be moved by placing the mouse pointer inside it and dragging 4 Change the frame size To change the frame size click a point inside the frame with the mouse pointer When square handles are displayed on the frame edges place the mouse pointer on one of them and drag it 5 Acquire images by clicking the XYZ XYT or XYZT button 6 After acquiring images select Normal from the list displaye...

Page 181: ...ire panel Then select Fast in the list shown below 512 by 512 is set automatically in the Size drop down list in the Scan Mode group box X2 is set and shown in gray out display automatically in the Zoom scale in the Pan group box 3 To change the zoom ratio use the Zoom scale in the Pan group box 4 Select any button to acquire image such as the XY Repeat button High speed image acquisition is avail...

Page 182: ...en select Sequen in the list shown below Selecting Sq Clp Sq Rct or Line Sq enables the image acquisition of the desired rectangle area or that of the rectangle area at desired angle or a line sequential scan Line Sequential Scan Ordinary sequential scan is performed for every one frame for every group whereas line sequential scan is performed for every one line for every group Therefore line sequ...

Page 183: ...ts with the smallest group number set To prevent unwanted color fading it is recommended to acquire the image from the reagents with longer wavelengths 5 Click the Seq Once button to acquire the image 6 When transmitted observation is also attempted the FLUOVIEW dialog box and the Lasers sub panel appear to specify the laser type which has not been specified yet Fig 2 24 FLUOVIEW Dialog Box Click ...

Page 184: ...n displayed in the pressed in condition 8 Click the OK button in the FLUOVIEW dialog box Image acquisition starts 9 Adjust the brightness with the PMT Offset and Gain LED sliders in the Ch group box in the Acquire panel See section 2 2 1 4 9 Adjusting the Image Brightness for details 10 After acquiring images select Normal from the list displayed below the Surface XY Norm option button in the Scan...

Page 185: ...rvation for operation procedure 2 Select Rect under the Surface XY option button in the Scan Mode group box in the Acquire panel A frame indicating the control range appears in the Live panel 3 Move the frame to the area to be observed To move the frame place the mouse pointer inside it and drag the mouse 4 Change the frame size Place the mouse pointer inside the frame and click to display square ...

Page 186: ...the frame can be varied according to the mouse pointer movement Place the mouse pointer on an inclining position and click the mouse left button to fix the frame inclination angle 7 Acquire the image with the buttons such as the XYZ XYT XYZT buttons 8 After acquiring the image select Normal under the Surface XY option button in the Scan Mode group box in the Acquire panel to return from the image ...

Page 187: ... Depth XZ option button in the Scan Mode group box in the Acquire panel A line indicating the control range appears in the Live panel 3 Move the line to the area to be observed To move the line place the mouse pointer on it and drag the mouse 4 Change the line length Place the mouse pointer on the line and click to display square handles on the two extremities of the line Then place the mouse poin...

Page 188: ... panel to return from the image acquisition mode 2 2 9 Display the change of image intensity Point Scan The change of fluorescence intensity according to time lapse can be displayed graphically by irradiating the laser to certain point on the image If you have FLUOVIEW TIME COURSE software image acquisition can be started with buttons keys or input of external trigger signals 1 Acquire an image in...

Page 189: ...ars in the Live panel 3 Move the cross cursor to the area you want to observe To move the cross cursor place the mouse pointer on it and drag the mouse 4 Set the scan speed for image acquisition Set the speed in the Scan speed group box of the Acquire panel Cross cursor Graph window Cross cursor Scan the center of the cross cursor ...

Page 190: ...the bottom right of the Acuire panel select the Time Series sub panel Set the time for measurement with the or button in the Scanning text box 6 Set the starting method for image acquisition If you have FLUOVIEW TIME COURSE software For setting procedure see FLUOVIEW TIME COURSE software user s manual 7 Click the Ptx T button to acquire the image The image at the coordinates set in the Live panel ...

Page 191: ...raph window can be modified by double clicking the mouse button on it When a certain area is specified by dragging the mouse from the top left to the bottom right on the graph while pressing down its left button the specified area can be magnified When the right button of the mouse is dragged on the graph the graph can be scrolled The magnification or scrolling of the graph can be canceled by drag...

Page 192: ...on the graph window the Editing dialog box appears and the graph parameters or the graph display method can be modified 8 After image acquisition select Normal under the Surface XY option button in the Scan Mode group box in the Acquire panel to return from the image acquisition mode TIP ...

Page 193: ...e section 2 2 1 Image Acquisition with XY observation 2 Select Line XT option button in the Scan Mode group box in the Acquire panel or Free under the Depth XZ option button A line indicating the scanning range appears in the Live panel Free under the Line XT option button is selected in the following Fig A line can be specified for one display 3 Move the curved line to the area you want to observ...

Page 194: ...dle The handle can be add by clicking the mouse on desired coordinates To delete the handle click the mouse on it Right click the mouse on the coordinates but the handles to fix the shape after the change 5 Set the scan speed for image acquisition Set the speed in the Scan speed group box of the Acquire panel The speed recommended for this scan mode are as follows 2µs The scroll bar indicates Fast...

Page 195: ...etc to acquire the image The image shown in the Live panel after image acquisition is the image that acquired at the curved line specified on the image and arranged uprightly in the X direction from the top to bottom The image shown after image acquisition is the same a width as that in the X direction specified in the Live panel Same as that in the XT observation mode 8 After image acquisition se...

Page 196: ...his section Three kinds of the laser excitation mode Disable REX and Bleach are selectable according to your purpose And each mode can be switched in the XYT observation In use of the REX or Bleach mode a setup of REX mask file is required Disable mode An ordinary mode to acquire an image of specified size REX mode The mode which sets the REX mask file to excite laser only to the target part and a...

Page 197: ...tion in the Line mode Normal Slant and Free can not be performed The specimen in the images carried in this section is not for FRAP but for the user s manual 2 2 11 1 Making REX Mask File 1 Display the Live panel at the front position 2 Select the Annotate button in the tool bar at the bottom left of the screen Annotate button NOTE NOTE ...

Page 198: ...Polyregion or Free region button And drag on the image to specify the region of laser excitation In order to specify two or more regions click the mouse on or inside the region with holding down the SHIFT key after a region is specified Circle button Free Region button Rectangular button Polyregion button Specify the region TIP ...

Page 199: ...Fig 2 26 Lasers sub panel When the Make REX mask button is displayed in gray click and select the specified region on the Live panel When two or more regions are specified select all the regions to be masked and let the handles be displayed around each region Handle Make REX mask button Makes the REX mask file of the specified region TIP ...

Page 200: ...panel has already been created on the FV300 software A panel is continuously created after REX_Live0 The REX mask file can be saved The saving procedure is completely the same as that of a display For details see section 2 3 1 2 Saving a Display in this manual Make REX mask button Make the REX mask file which masks the region outside of the specified region TIP TIP ...

Page 201: ...ensity dialog box The laser intensity of the REX mask file is setup on percentage to 100 The region becomes brighter as the value is close to 100 When the laser intensity has been set to other than 100 in the Laser Intensity group box in the Lasers sub panel the value can be decreased in the Laser Intensity dialog box However the laser intensity value does not equal to the value obtained by multip...

Page 202: ...e square power of zoom magnification per time can be secured Moreover using the objective of larger NA concentrates the laser irradiation spot in Z direction and stronger laser can be irradiated Therefore a preliminary experiment is required to set up the laser intensity the objective the zoom ration of the REX mask file according to a specimen 1 Preliminary Experiment 1 Set the laser intensity th...

Page 203: ...r acquiring an image 3 Making REX Mask File See section 2 2 11 1 Making REX Mask File for the procedure to make the REX mask file If the setup of the region of laser excitation remains after making the REX mask file the intensity change of the region can be observed in the TIME COURSE software optional See the FV TIEMPO TIME COURSE software User s manual for details of the TIME COURSE software ...

Page 204: ...menu as shown below Select the image to be masked in the menu In order to use the already saved file as a REX mask file open the image beforehand The opening method of the REX mask file is completely the same as that of an image For details see section 2 3 2 Opening Previously Saved Images in this manual 3 The icon of the selected REX mask file is displayed inside the frame The REX mask file displ...

Page 205: ...is completely the same as that of an image For details see section 2 3 2 Opening Previously Saved Images in this manual 6 The icon of the selected REX mask file is displayed inside the frame 7 Set each laser ON OFF and their intensity If necessary use the Laser Intensity dialog box in the Lasers sub panel to set the laser ON OFF And the value set in the Laser Intensity dialog box can be obtained a...

Page 206: ...OPERATIONS Image Acquisition 2 96 Page 5 Setting the XYT Observation mode 1 Confirm that XY Norm is displayed in the Scan Mode group box in the Acquire panel 2 Select the XYT option button in the Acquire panel ...

Page 207: ...acquisition is set to 5 minutes or 300 seconds in the Total Time text box The time for image acquisition differs according to the interval and the number of scans 6 Setting the TIME COURSE Software optional Setting of the real time graph is required when you use the TIME COURSE software See the FV TIEMPO TIME COURSE software User s manual for details of the TIME COURSE software Rest Time text box ...

Page 208: ...e is effective to prevent fading of the obtained data 1 Select the Disabled option button in the AOTF group box in the Lasers sub panel 2 Click the XYT button to start image acquisition 3 Select the Bleach mode option button in the AOTF group box in the Lasers sub panel Select the Bleach mode option button within 10 seconds before the next scanning is started In the TIME COURSE software optional w...

Page 209: ...he Disabled or REX mode option button within 10 seconds before the next scanning is started In the TIME COURSE software optional when the REX mask file in the REX or Bleach mode is changed while image scanning is performed obtaining the real time graph the laser is irradiated while the pop up menu is displayed however the real time graph is not plotted Please be aware this before using The laser o...

Page 210: ...cquired When performing the forth or subsequent scan be sure to switch the REX mode and Bleach mode between scans In the TIME COURSE software optional when the REX mask file in the REX or Bleach mode is changed while image scanning is performed obtaining the real time graph the laser is irradiated while the pop up menu is displayed however the real time graph is not plotted Please be aware this be...

Page 211: ... select one of them Display panel Displays the image The image file name is shown in the panel page tab File Type drop down list Selects the type of the files to be displayed in the Files list box Directory list box Shows the directories in the hierarchical structure Double click the required directory If the required directory is not shown double click the upper level of the current directory or ...

Page 212: ...layed in the Icon field in the Files list box In all observation modes the icons for 3 or more channels are identical Image Icon Significance XZ observation XZ observation 2 channel mode Xt observation Xt observation 2 channel mode XZT observation XZT observation 2 channel mode XY observation XY observation 2 channel mode XYt observation XYt observation 2 channel mode XYZ observation XYZ observati...

Page 213: ...modes Y Image sizes in the Y direction X Image sizes in the X direction T Number of images acquired over time Ch Number of image channels Date Image saving dates An Number of images created when constructing a stereo image using the Visualize function Size Image file sizes Stx 2 is displayed when stereo images stereo 3D image or stereo image to be viewed through color eyeglasses are constructed an...

Page 214: ...on Also set the save end position in the similar way 3 When the images to be saved are acquired from more than one channel select whether saving images from more than one channel simultaneously or only a single image from a single channel Use the Display channel switch buttons for this selection The images will be saved according to the conditions of the selected channels Example When only the ima...

Page 215: ... File Name text box 8 Click the Save button If a file with the same name as the entered file name already exists a dialog box is displayed to ask if you want to overwrite the existing file If you do not want to overwrite it click the NO button and enter another file name NOTE One Point The Save Experiment As dialog box can also be displayed by a mouse operation 1 Display the image to be saved at t...

Page 216: ...image to be saved display the image to be saved using the Display buttons 3 Click the Display button in the Save group box The Save Display dialog box as shown below appears Fig 2 30 Save Display Dialog Box 4 When it is required to save the comment drawn on the image together with the image check the Include Overlay check box 5 Click the Display button in the Save group box The Save Experiment As ...

Page 217: ... the File Name text box 9 Click the Save button If a file with the same name as the entered file name already exists a dialog box is displayed to ask if you want to overwrite the existing file If you do not want to overwrite it click the NO button and enter another file name NOTE One Point The Save Display dialog box can also be displayed by a mouse operation 1 Display the image to be saved at the...

Page 218: ... are saved together or the image slice of only 1 channel is saved Use the display switching button for the selection The image will be saved in the channel condition as displayed For the channel switching see section 2 4 3 Switching the Displayed Channels 3 Specify a desired area in the image This operation is not necessary if an area has already been specified Click the Annotate button in the too...

Page 219: ...ist of buttons disappear 7 Click the Experiment button in the Save group box The Save Experiment As dialog box appears Fig 2 32 Save Experiment As Dialog Box 8 Use the Save In drop down list if you want to change the save destination drive and directory 9 Select FLUOVIEW Multi Tiff in the Save as Type drop down list It is not possible to save only the specified area of image in the Single TIF BMP ...

Page 220: ... dialog box as shown below appears Fig 2 33 Saving a subset of Experiment s Data Dialog Box The Saving a subset of Experiment s Data dialog box does not appear if no area is specified in the image on the Display panel 13 Click the Yes button Size of the specified area TIP ...

Page 221: ...isplay Speed 3 Select whether saving images from multiple channels simultaneously or only an image from a channel if the images to be saved are acquired from multiple channels Use the Display Channel Switch button for this selection The image is saved according to the condition of the selected channel For the channel switching see section 2 4 3 Switching the Displayed Channels 4 Click the Animatio...

Page 222: ...2 3 1 1 Saving Images As a Series for the operation method 1 Click the Experiment button in the Save group box 2 Select the file type from the Save as Type drop down list in the Save Experiment As dialog box Three file types are available as detailed below Fluoview Multi Tiff tif TIFF format designed for use with FLUOVIEW Used for image analysis processing etc on FLUOVIEW Single TIF s 8 bit tif Si...

Page 223: ... comment drawn on it as an image select the file type according to the usage of the image as described below Fluoview Multi Tiff Select when the image will be analyzed or processed after save Opening the image also opens the comment which can be changed or measured as required 24 bit 8bit file type Select when the image will be handed to another application The following table shows the list of im...

Page 224: ...e of only Ch3 is saved Save file name abcdefgh tif Ch3 Nos 0 to 3 The saved image contains the image slices of the channels used in the above views The images of Ch1 Ch2 and Ch3 are saved Save file name abcdefgh tif Ch1 Ch2 Ch3 0 3 The images of Ch1 Ch2 and Ch3 are saved Save file name abcdefgh tif Ch1 Ch2 Ch3 Nos 0 to 3 The image is saved in the selected channel condition Note that a file does no...

Page 225: ... in the above views 4 images are saved from each of Ch1 Ch2 and Ch3 Save file names abcdefghV0 tif Ch1 No 0 abcdefghV2 tif Ch3 No 0 abcdefghV3 tif Ch1 No 1 abcdefghV5 tif Ch3 No 1 abcdefghV6 tif Ch1 No 2 abcdefghV8 tif Ch3 No 2 abcdefghV9 tif Ch1 No 3 abcdefghV11 tif Ch3 No 3 4 images obtained by merging Ch1 Ch2 and Ch3 images are saved Display gradation of the images are poor than original Save f...

Page 226: ...0 abcdefghV2 tif Ch3 No 0 abcdefghV3 tif Ch1 No 1 abcdefghV5 tif Ch3 No 1 abcdefghV6 tif Ch1 No 2 abcdefghV8 tif Ch3 No 2 abcdefghV9 tif Ch1 No 3 abcdefghV11 tif Ch3 No 3 4 images obtained by merging Ch1 Ch2 and Ch3 images are saved Save file names abcdefgh0 tif Ch1 Ch2 Ch3 No 0 abcdefgh3 tif Ch1 Ch2 Ch3 No 3 The relationship between the saved channels and files are variable depending on the condi...

Page 227: ... the above views 4 images are saved from each of Ch1 Ch2 and Ch3 Save file names abcdefghV0 tif Ch1 No 0 abcdefghV2 tif Ch3 No 0 abcdefghV3 tif Ch1 No 1 abcdefghV5 tif Ch3 No 1 abcdefghV6 tif Ch1 No 2 abcdefghV8 tif Ch3 No 2 abcdefghV9 tif Ch1 No 3 abcdefghV11 tif Ch3 No 3 4 images obtained by merging Ch1 Ch2 and Ch3 images are saved Display gradation of the images are poor than original Save file...

Page 228: ...cdefghV2 tif Ch3 No 0 abcdefghV3 tif Ch1 No 1 abcdefghV5 tif Ch3 No 1 abcdefghV6 tif Ch1 No 2 abcdefghV8 tif Ch3 No 2 abcdefghV9 tif Ch1 No 3 abcdefghV11 tif Ch3 No 3 4 images obtained by merging Ch1 Ch2 and Ch3 images are saved Save file names abcdefgh0 bmp Ch1 Ch2 Ch3 No 0 abcdefgh3 bmp Ch1 Ch2 Ch3 No 3 The relationship between the saved channels and files are variable depending on the condition...

Page 229: ...e Perform the same operations as steps 1 and 2 above before the following Place the mouse pointer on the desired image file name in the Files list box and drag the file name to the Open frame on the upper part of the File I O panel The mouse pointer transforms to an image icon during dragging Place the mouse pointer on the desired image file name in the Files list box and double click it 2 3 3 Shr...

Page 230: ...se pointer transforms to an image icon during dragging 3 Click the Done button in the Experiments in Memory dialog box to close it Multiple images displayed can be shredded simultaneously 1 Display the Experiments in Memory dialog box while showing multiple images in the Display panel 2 Pressing down the Shift key select multiple image files to be shredded 3 Click the Shredder button 4 Click the D...

Page 231: ...anel The Experiments in Memory dialog box appears as shown below Fig 2 36 Experiments in Memory Dialog Box 2 In the Experiments in Memory dialog box select the file name of the image to be saved with comment and click the Comments button The Image Comments dialog box appears as shown below Fig 2 37 Image Comments Dialog Box 3 Display the Comments panel at the front Experiment List button ...

Page 232: ...s panel is saved simply but it is not yet saved in the image file To save the comment in the image file use the Save button in the File I O panel 6 Click the OK button 7 Click the Done button in the Experiments in Memory dialog box to close it 8 Display the File I O panel at the front 9 Click the Experiment button in the Save group box See section 2 3 1 Saving Images for details NOTE NOTE TIP One ...

Page 233: ...eters and click the Comments button 3 Display the Image Attributes panel at the front The panel as shown below appears where the information on the selected image is shown Fig 2 39 Image Attributes Panel Experiment List button Intensity Shows the length of the data expressing the intensity value CH Shows the number of image acquisition channels Z Displays number of Z slices Z Shows the number of s...

Page 234: ...the number of steps in the Z direction or the objective setting if it is required to restore the previous setting click the Restore Attributes button However once the Set Attributes button is pressed the original setting cannot be restored The objective setting can be changed even after having acquired the image If the objective is not set with the Acquire panel before image acquisition the analys...

Page 235: ...n the right The kind of information displayed when each item is selected is shown below 6 After checking the information click the OK button 7 Click the Done button in the Experiments in Memory dialog box to close it Range of intensity values mapped for each channel Changes contents of Image Field LUT status Acquisition parameters Version information Comment ...

Page 236: ... Point The Image Comments dialog box can also be displayed by a mouse operation 1 Display the image to be saved at the front of the Display panel and right click a point in the image 2 A pop up menu as shown below is displayed 3 Select Experiment Properties from the menu ...

Page 237: ...vation condition can be saved as ASCII text file in a disk 1 Display the Display panel of the image to be saved at the front position 2 Select the Experiment List button in the tool bar displayed at the bottom of the File I O panel The Experiments in Memory dialog box as shown below appears Fig 2 41 Experiments in Memory dialog box Experiment List button ...

Page 238: ... Save to txt dialog box The LUT Ch1 to LUT Ch6 check boxes are displayed according to the channel used for image acquisition 5 Check the check box of the item to be saved Properties button in the Export to File group box Saves the image information and observation condition Saves the range of intensity mapped to every channel Save button Saves the checked item into the text file Cancel button Canc...

Page 239: ... text txt is selected in the Save as type drop down list 9 Enter a file name into the File Name text box 10 Click the Save button When the text file having the same name as the entered name is already exists the dialog box appears to ask you to overwrite the existing file or not When overwriting is not required click the NO button and enter another name to save the file When the image to be saved ...

Page 240: ...ved in a file and they can also be read 2 3 7 1 Saving the Region File 1 Display the Display panel of the image including the region to be saved at the front position 2 Select the Experiment List button in the tool bar at the bottom of the File I O panel The Experiments in Memory dialog box as shown below appears Fig 2 45 Experiments in Memory dialog box Experiment List button ...

Page 241: ... box 4 Select the Annotations ROIs button in the Export to File group box The Save Experiment As dialog box as shown below appears Fig 2 47 Save Experiment As dialog box 5 Select the save destination drive or folder in the Look in drop down list 6 Select FLUOVIEW ROI roi in the Files of type drop down list Annotations ROIs button in the Import from File group box Opens the file saving the region A...

Page 242: ... name to save the file 2 3 7 2 Reading the Region File 1 Display the Display panel of the image including the region to be read at the front position 2 Select the Experiment List button in the tool bar at the bottom of the File I O panel The Experiments in Memory dialog box as shown below appears Fig 2 48 Experiments in Memory dialog box 3 In the list in the Experiments in Memory dialog box select...

Page 243: ... box 5 Select the drive or folder where the file to be read is saved in the Look in drop down list 6 In the list below the Look in drop down list double click the folder or sub folder where the data to be read is saved to open 7 Select FLUOVIEW ROI roi in the Files of type drop down list Annotations ROIs button in the Export to File group box Saves the region into a file Annotations ROIs button in...

Page 244: ...le including the region to be read and that of the specified file differ the Careful dialog box as shown below appears Clicking the OK button reads the file in different image size whereas clicking the No button cancels reading the region file When the region file is read in different image size the upper left of the image is assumed as reference TIP ...

Page 245: ...was acquired from more than one channel select the channels to be colored using the Ch1 Ch2 Ch3 Ch4 and or Ch5 option buttons The Ch1 Ch2 and Ch3 option buttons are displayed only when an image acquired from more than one channel mode is displayed selected 4 From the Standard Color LUTs group box select the desired color button The selected LUT will be applied immediately to the image in the Displ...

Page 246: ...og box can also be displayed by a mouse operation 1 Display the image to be colored at the front of the Display panel and right click a point in the image 2 A pop up menu as shown below is displayed 3 Select View Processor from the menu then select Intensity Scaling from the displayed sub menu ...

Page 247: ...ck boxes below the graph in the Color LUT Tool group box 5 Set the range of intensity graph application using the Low and High scales in the Intensity Mapping group box Double clicked the Low scale High scale and Gamma text box they are reset to the default values 0 with the Low scale 4095 or 255 with the High scale and 1 0 with the Gamma text box 6 The inclination of the graph in the Color LUT To...

Page 248: ...the OK button to exit from the LUT editing One Point The Color Tool dialog box can also be displayed by a mouse operation 1 Display the image to be colored at the front of the Display panel and right click a point in the image 2 A pop up menu as shown below is displayed 3 Select View Processor from the menu then select Intensity Scaling from the displayed sub menu ...

Page 249: ...ty graph application with the Low and High scales in the Intensity Mapping group box Dragging one end of the graph makes it possible to change the inclination The set intensity graph is immediately reflected in the image in the Display panel Double clicked the Low scale High scale and Gamma text box they are reset to the default values 0 with the Low scale 4095 or 255 with the High scale and 1 0 w...

Page 250: ...a previously saved LUT click the Load LUT button 2 4 3 Switching the Displayed Channels Ch1 Ch5 The buttons on the bottom left of the screen can be used to select where the image of a single channel or images of multiple channels are to be displayed For the simultaneous display of multi channel images see section 2 4 4 Displaying Images of Multiple Channels Simultaneously TIP ...

Page 251: ... 54 Panel Displaying the Channel 2 Image 2 4 4 Displaying Images of Multiple Channels Simultaneously Side By Side Views Over And Under Views Single View Images from multiple channels can be displayed either by merging them or placing them side by side It is also possible to display the image of only one of these channels Use the buttons displayed at the top of the Display panel and those on the bo...

Page 252: ...g button at the top of the Display panel The list of buttons as shown below appears 3 From the displayed list of buttons click one of the Side by side Views button or Over and under views button The icon shown in the Display switching button will change to the icons of the Side by side views button or Over and under views button Fig 2 55 Panel Displaying Images of Two Channels Side By Side Display...

Page 253: ...w the images of multiple channels side by side At the same time the icon shown in the Display switching button will change to the icon of the Single View button 4 Click the image to display the Display channel switch buttons at the bottom left of the image 5 Select the channels which should be displayed by pushing Display channel switch buttons The color of a selected channel s button becomes dark...

Page 254: ...usly can be changed Increased image is only to be displayed The image increased in Add View is not subjected to these operations described below 2 4 5 1 Increasing the Number of Divided Images 1 Display the Display panel of the image to be changed at the front Fig 2 57 Display panel 2 Right click the image A pop up menu as shown below appears TIP ...

Page 255: ...mage Display Method 2 145 Page 3 Select Views then select Add a view in the sub menu 4 A view is added to the rightmost position on the Display panel Fig 2 58 Display Panel After View Addition Up to 6 views can be displayed at once TIP ...

Page 256: ...e 2 4 5 2 Decreasing the Number of Divided Images 1 Display the Display panel of the image to be changed at the front Fig 2 59 Display panel 2 Right click the image A pop up menu as shown below appears 3 Select Views then select Remove a last view in the sub menu ...

Page 257: ...APPLIED OPERATIONS Changing the Image Display Method 2 147 Page 4 A view is removed from the Display panel Fig 2 60 Display panel after View Removal ...

Page 258: ...rom the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame Display button Press to start display of n slices of image from the end position to the start position sequentially successive di...

Page 259: ...how the images are displayed in the Display panel Columns text box Sets the number of columns or the number of image slices displayed in a horizontal row Experiment Shows the acquisition parameters used in acquisition of the displayed images Icon of the displayed images image slices shown together Tile Over drop down list Select the acquisition parameter to be based on when arranging the images Se...

Page 260: ...oxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box 3 When there are multiple images to be displayed select the following items in the Tile Over drop down list Self The same images as the image being displayed will be displayed Z Images are displayed according to change in multiple sections T Images are displayed according to change in ti...

Page 261: ...APPLIED OPERATIONS Changing the Image Display Method 2 151 Page Fig 2 62 Panel Displaying Images Per Channel ...

Page 262: ...gether for simultaneous view The operation method is identical to the method for displaying images per channel except for the following point See section 2 4 7 1 Displaying Multiple Images Per Channel z With images acquired in a multi channel mode select the display method from the Display Type drop down list Fig 2 63 Panel Displaying Images by Overlaying Different Channels ...

Page 263: ...ns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of the Tiling group box 3 Select T from the Tile Over drop down list 4 When the time lapse images were acquired in a multi channel mode select the display method from the Display Type drop down list 5 Click the New Page button A new Display panel appears showing the images displayed per channe...

Page 264: ...splaying Same Images in Different Display Methods Images composed of multiple image slices can be displayed in more than one display methods together 1 Display the Display panel of one of the images image composed of multiple slices to be displayed together The icon of the image is displayed in the frame at the top left of the Tile panel and the acquisition parameters used in image acquisition are...

Page 265: ...e of the displayed images Buttons appear above the clicked image 6 Switch the image display using the displayed buttons For details on the display switching method see sections 2 4 3 Switching the Displayed Channels 2 4 4 Displaying Images of Multiple Channels Simultaneously and 2 4 6 Switching the Display Method of Multiple Images 7 Perform steps 5 and 6 above for each of the displayed images Fig...

Page 266: ...le or New Page button All of the images in the panel are re displayed using the same display method as that set in step 2 above 2 4 7 7 Displaying Different Images Together Two completely different images can be displayed together The two images to be displayed together should be acquired by observation or loaded by opening a file If two images are not available prepare them by image acquisition o...

Page 267: ... in the image acquisition are displayed in the Experiment panel The mouse pointer turns into the image icon during dragging 4 Click the Done button in the Experiments in Memory dialog box to close it 5 Set the number of images to be displayed together by using the and buttons in the Columns and Rows text boxes How the images will be arranged can be confirmed in the gray box at the upper part of th...

Page 268: ...ars showing the two images one above the other The image of the file displayed in the frame at the top left of the Tile panel is displayed on the upper part of the Display panel and that of the file displayed in the frame at the top right is displayed on the lower part Use the Retile button when it is required to re arrange the images in the currently displayed Display panel Fig 2 68 Panel Display...

Page 269: ... of the image to be displayed 2 Click the Full Screen button in the toolbar at the bottom left of the screen When the mouse left button is clicked while the image is displayed full screen the buttons which are usually displayed on the top of the Display panel Display buttons etc and those usually displayed on the bottom right Display channel switch button can be displayed in the image Fig 2 69 Pan...

Page 270: ...nt The image can also be displayed full screen by the mouse operation on the image 1 Display the Display panel of the image to be displayed full screen and click the mouse right button on the image 2 A pop up menu as shown below appears 3 Select FullScreen Display from the ...

Page 271: ...low are displayed on the top of the Display panel Usually the Auto button is displayed and clicking it displays the list of buttons shown below Us these buttons to magnify or reduce the image Fig 2 70 Panel Showing a 2 1 Magnified Image AUTO button Magnifies or reduces the image according to the current Display panel size 2 1 button Magnifies the original image by 2 times 1 1 button Displays the i...

Page 272: ... images 1 Display the Filters sub panel in the Process panel at the front Fig 2 71 Filters Sub panel Icon of the image being displayed Image to be processed by filtering Smoothing Filters group box Provides a command button for each noise filter Sharpening Filters group box Provides a command button for each of the contour enhancement and image sharpening filters DIC Image Filter group box Used fo...

Page 273: ...image slices the range of image slices to be filtered can be specified using the Set start position and Set end position buttons above the image First display the image slice to start filtering using the Display button and click the Set start position button Then set the image slice to end filtering in the same way as above 3 When the image to be filtered was acquired in the multi channel mode fil...

Page 274: ...ns If the image contains noise the noise is also enhanced It has two filter formats X and Y as shown below The format providing the larger value after filtering is used 1 2 1 1 0 1 0 0 0 2 0 2 1 2 1 1 0 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter z Click the Sobel button Sobel button ...

Page 275: ...tering except for the following point See section 2 5 1 1 1 Laplacian filter z Click the Highpass X button 4 Highpass Y filter The HIGH PASS Y filter passes the high frequency structures in the Y direction of image In this way it can extract details by detecting positions with large variation This processing is useful for making structures clear or extract the edges The filter format is as shown b...

Page 276: ...Reduction When random noise interferes with an image its irregularity increases and it become harder to see Such noise can be reduced by means of filtering Three kinds of filters are available for noise reduction 1 Averaging filter The averaging filter is used to eliminate details in image or reduce noise However as it makes everything in an image smooth it also makes the edge sections dull which ...

Page 277: ...y structures In this way it can eliminate small grains and provide smooth noise reduced image filter format is as shown below 1 2 1 2 4 2 1 2 1 The operation method is identical to Laplacian filtering except for the following point See section 2 5 1 1 1 Laplacian filter z Click the Lowpass button 2 5 1 3 Image Sharpening 1 Sharpen filter The sharpen filter turns blurred image into a clear image Th...

Page 278: ... Display the Filters sub panel in the Process panel Fig 2 73 Filters Sub panel 2 Enter the appropriate correction factor for the image background in the kg text box The correction factor can be set between 0 1 and 1 in steps of 0 1 The standard factor is 1 If the effect is extreme try using 0 5 Irregularities in brightness are observed across the diagonal line of the background DIC leveling button...

Page 279: ...d in the Display panel and shows an image after the correction of the DIC level irregularities of the background Fig 2 74 Level Panel After DIC Level Correction 4 If the DIC level irregularities cannot be reduced repeat steps 1 to 3 above by varying the correction factor The background irregularities are solved ...

Page 280: ...ges the contrast by noticing a certain section between 0 and 4095 and mapping this section to values between 0 and 255 1 Display the Process panel at the front 2 Display the Histogram sub panel of the Process panel at the front Fig 2 75 Histogram Sub panel 3 Display the Display panel of the image you want to change the contrast The icon of the image is displayed in the frame at the top left of the...

Page 281: ...he target channel s use the Display channel switch buttons The histogram of the selected channel s is displayed Example When only the Ch1 image is displayed the histogram of Ch1 is displayed and mapping of only the Ch1 image is possible For the switching of channels see section 2 4 3 Switching the Display Channels 5 Click the Compute Histogram button A histogram appears as shown below Fig 2 76 His...

Page 282: ...l mode select the channel s to be subjected to LUT intensity mapping re assignment using the Channel option buttons 9 Select the region to be subjected to mapping re assignment using the scale in the Selected View field While moving the scale confirm the change in contrast in the Display panel The intensity values in the selected region are mapped re assigned to intensity values from 0 to 255 and ...

Page 283: ...Display the Math sub panel of the Process panel Fig 2 78 Math Sub panel Scalar Operations group box Provides the command buttons for use in operations between image and constant Sets the first image of the operation The icon of the image is displayed here Sets the second image of the operation The icon of the image is displayed here This image needs not be set in operations between image and const...

Page 284: ...nel Before the dragging and dropping the frame at the top left shows the icon of the image file displayed in the Display panel The mouse pointer turns into the image icon during dragging 4 From the Experiments in Memory dialog box select the file name of the second file and drag it to the frame at the top right of the Process panel The icon of the image is displayed in the frame at the top right o...

Page 285: ...he Add 2 Images button in the Multi Image Operations group box A new Display panel showing Image Image in the page tab appears displaying the image obtained by the addition operation To add a constant to an image Click the Add to Image button in the Scalar Operations dialog box A new Display panel showing Image Const in the page tab appears displaying the image obtained by the addition operation F...

Page 286: ...n image by an image image by a constant is possible as described below The operation method is identical to Image Image Image Constant except for the following point See section 2 5 3 1 Image Addition z To multiply an image by an image Click the Multiply 2 Images button in the Multi Image Operations group box A new Display panel showing Image Image in the page tab appears displaying the image obta...

Page 287: ...e page tab appears displaying the image obtained by the division operation 2 5 3 5 NOT Image The NOT operation of an image allows the bright and dark areas of the image to be reversed 1 Display the Logical sub panel of the Process panel at the front Fig 2 81 Logical Sub panel 2 Display the Display panel of the image to be subjected to NOT operation at the front The icon of the image is displayed i...

Page 288: ...ERATIONS Image Processing 2 178 Page 3 Click the NOT Image button A new Display panel showing ImageNot in the page tab appears showing the image obtained by the operation Fig 2 82 ImageNot Panel NOT Image button ...

Page 289: ... of the Process panel The Experiments in Memory dialog box appears as shown below Fig 2 84 Experiments in Memory Dialog Box Experiment List button Image AND Image button Executes an AND operation Sets the image to be subjected to operation The icon of the image is displayed here Sets the second image of the operation The icon of the image is displayed here Image XOR Image button Executes an exclus...

Page 290: ... at the top left shows the icon of the image file displayed in the Display panel The mouse pointer turns into the image icon during dragging 4 From the Experiments in Memory dialog box select the file name of the second file and drag it to the frame at the top right of the Process panel The icon of the image is displayed in the frame at the top right of the Process panel The mouse pointer turns in...

Page 291: ...ND Image Panel 2 5 3 7 Image OR Image Two different images can be ORed The operation method is identical to the AND operation between two different images except for the following point See section 2 5 3 6 Image AND Image z Click the Image OR Image button A new Display panel showing Image OR Image in the page tab appears showing the image obtained by the operation Image AND Image button Image OR I...

Page 292: ...ration method is identical to the AND operation between two different images except for the following point See section 2 5 3 6 Image AND Image z Click the Image XOR Image button A new Display panel showing Image XOR Image in the page tab appears showing the image obtained by the operation Image XOR Image button ...

Page 293: ...icking this field displays the Enhanced Profile Plot window When a region is specified the bird s eye view is displayed and double clicking this field displays the Intensity Map window Measurement Results box Shows the measurement data of the specified line or region Region Histogram box Shows the histogram of the specified line or region Double clicking this field displays the Enhanced Histogram ...

Page 294: ...rs as shown below 4 From the displayed buttons click the Line button Poly Line button or Free Line button 5 Specify the straight line polygonal line or free line on the image in the Display panel They can be specified as described below z To specify a straight line On the image place the mouse pointer on the point you want to start the straight line and drag until the point you want to end it z To...

Page 295: ...annot be displayed when the handles are not displayed The checked line can be moved deleted or changed of size or color This is possible with the same method as entering comment in the image For details see sections 2 11 6 2 11 7 2 11 8 and 2 11 9 in section 2 11 Entering Comment in Image 6 Click the Annotate button so that the list of buttons disappears 7 Click the Begin Analysis button The inten...

Page 296: ...to the right of the magnified graph When the mouse pointer is placed on a graph line while the Ctrl or Alt key is held depressed the coordinates can be displayed The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application for details Save button Saves the profile data in a file using an Excel compatible format Copy button Copies the plotted ima...

Page 297: ...hannels are analyzed simultaneously or only one channel is analyzed To select the target channel s use the Display channel switch buttons Only the channel s being displayed will be analyzed Example When only the Ch1 image is displayed only the Ch1 image is analyzed For the switching of channels see section 2 4 3 Switching the Display Channels 4 Click the Annotate button in the toolbar at the botto...

Page 298: ...f the polygon to be checked After clicking the last corner point click the right button of the mouse to connect the last clicked point to the first clicked point z To specify a free region On the image specify a region by dragging Then release the mouse button to complete dragging The point where the dragging was ended will be connected to the point where it was started A region is displayed on th...

Page 299: ...so that the list of buttons disappears 8 Click the Begin Analysis button The bird s eye view of the specified region will be displayed in the Intensity Profile box of the Analyze panel The Measurement Results box in the Single sub panel shows the measurement results such as the area Area horizontal and vertical lengths X Y Z or T and perimeter length Perimeter of the region and the statistic resul...

Page 300: ...hannel mode the channel s to be subjected to the bird s eye view display can be selected using the Channel option buttons Fig 2 89 Panel After Analysis Region Specification Shows Measurement results including Perimeter Area X Y Z or T Statistical channel data including Total Average Std Dev ...

Page 301: ...ormat Copy button Copies the plotted image in the clipboard Close button Quits the Enhanced Profile Plot window and returns to the Analyze Tilt scale Sets the angle in the vertical direction The result can be confirmed with the small bird s eye view in the frame on the top left Angle scale Sets the angle in the horizontal direction The result can be confirmed with the small bird s eye view in the ...

Page 302: ...ows appears as shown below Fig 2 91 Enhanced Histogram Plot Window Line Specification When a desired area is specified by dragging the left button of the mouse on the graph the specified area can be magnified When the right button of the mouse is dragged on the graph the graph can be scrolled Save button Saves the profile data in a file using an Excel compatible format Copy button Copies the plott...

Page 303: ...d The histogram on a region in an image can be displayed The operation method is identical to displaying the intensity profile on a line See section 2 6 1 1 Intensity Values on a Line Line Profile Double click the Region Histogram window The Enhanced Histogram Plot windows appears as shown below Fig 2 92 Enhanced Histogram Plot Window Line Specification Save button Saves the profile data in a file...

Page 304: ... be scrolled The magnification or scrolling of the graph can be canceled by dragging the left button of the mouse from the bottom left to the right of the magnified graph When the mouse pointer is placed on a graph line while the Ctrl or Alt key is held depressed the coordinates can be displayed The displayed data can be applied to other applications See section 2 10 Transferring Data to Another A...

Page 305: ...ring the perimeter of a region is identical to that for displaying the bird s eye view of a region See section 2 6 1 2 Intensity Values on a Planar Region Bird s Eye View The measurement results can be written in comment by copying and pasting them in the Image Comments dialog box in the Experiments in Memory dialog box See section 2 3 4 Saving Comment Together with Image for details 2 6 3 2 Area ...

Page 306: ...ingle Slice Integration group box appears as shown below Select the direction of interest X or Y using the option buttons When the selected image was acquired in the XY observation mode the Single Slice Integration group box shows the option buttons for selection of the X or Y direction When the selected image was acquired in the XT observation mode the option buttons for selection of the X or T d...

Page 307: ...tion Z time lapse T to be subjected to the operation can be selected using the XYZ series and XYT series buttons above the image 4 When the image was acquired in the multi channel mode select whether the multiple channels are operated simultaneously or only one channel is operated To select the target channel s use the Display channel switch buttons Only the channel s being displayed will be analy...

Page 308: ...6 1 2 Intensity Values on a Planar Region Bird s Eye View A region is displayed on the image together with handles on the perimeter The region is selected as the target of mean value computation operation while the handles are displayed If the mouse is clicked in other place than inside the region specified on the image the handles will disappear On the contrary clicking the mouse in a region disp...

Page 309: ... and display their operation results together First specify the regions by repeating steps 7 and 8 above for each Use different colors for the regions After having set the regions click the mouse in the first region With the second regions and after click the mouse while pressing the Shift key depressed 12 Click the Annotate button so that the list of buttons disappears 13 Using the scale in the T...

Page 310: ...e Mean Intensity box The colors of the chart lines corresponding to the colors assigned to the regions When the image was acquired in the multi channel mode the channel number is displayed to the right of each chart line Fig 2 94 Panel After Analysis 15 Double click the Mean Intensity box The Average Intensity Trace window appears as shown below NOTE NOTE ...

Page 311: ...use pointer is placed on a graph line while the Ctrl or Alt key is held depressed the coordinates can be displayed The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application for details Save button Saves the profile data in a file using an Excel compatible format Copy button Copies the plotted image in the clipboard Close button Quits the Aver...

Page 312: ...Integrity Intensity Trace window appears as shown below Fig 2 96 Integrated Intensity Trace Window When a desired area is specified by dragging the left button of the mouse on the graph the specified area can be magnified When the right button of the mouse is dragged on the graph the graph can be scrolled Copy button Copies the plotted image in the clipboard Close button Quits the Integrated Inten...

Page 313: ... of the mouse from the bottom left to the right of the magnified graph When the mouse pointer is placed on a graph line while the Ctrl or Alt key is held depressed the coordinates can be displayed The displayed data can be applied to other applications See section 2 10 Transferring Data to Another Application for details TIP TIP TIP ...

Page 314: ...the XYZ multiple sections image 2 The following button is displayed at the top of the Display panel Usually only the XYZ series button is displayed When it is clicked a list of buttons appears as shown below 3 Click the Extend button 4 Click the Display button at the top of the Display panel repeatedly to build the extended focus image The extended focus image can also be displayed by clicking and...

Page 315: ...RATIONS Building an Image from a Different Viewpoint 2 205 Page Image No 0 Image No 1 Image No 2 Image No 3 Fig 2 97 Four Images Used in Building Extended Focus Image Fig 2 98 Panel Showing an Extended Focus Image ...

Page 316: ... end image by using the Frame advance or Successive display buttons at the top of the Display panel 4 Click the Set end position button at the top of the Display panel The Set start position and Set end position buttons are valid in the pushed in condition To cancel a previously set start or end position click the Set start position or Set end position button again When it is required to analyze t...

Page 317: ...anel of the XYZ multiple sections image 2 Click the Extended Focus View option button in the Rendered View group box 3 Click the Begin Visualize button to start the image building When it completes the built image is displayed in the Extended panel Display panel Shows the image The file name of the image is shown in the page tab of the panel Begin Visualize button Starts building the extended focu...

Page 318: ...APPLIED OPERATIONS Building an Image from a Different Viewpoint 2 208 Page Fig 2 100 Panel Showing Extended Focus Image ...

Page 319: ...xtended Focus View option button in the Rendered View group box 3 Click the Begin Visualize button to start the image building When it completes the built image is displayed in the Extended panel Fig 2 101 Panel Showing Extended Focus Image 2 7 2 Building line images to be viewed in Z direction The images acquired by cutting the XYZ image off vertically and horizontally can be displayed with the i...

Page 320: ... XY direction image displayed left above Make Multi Plane View button Display button Image display in XY direction The line image to be displayed in XZ or YZ direction can be moved by dragging the red line XZ or brown line YZ on the XY direction image Line image display in YZ direction Display the image of the line specified on the XY direction image Line image display in XZ direction Display the ...

Page 321: ...es not need to be changed Begin Visualize button Starts building the images for 3D display Initial view option button Sets the angle at which the rotation should start The angle itself can be set using the Initial Rotation Angle scale immediately below the option button Sequence views option button Sets the rotation angle per rotation step The angle itself can be set using the Rotation Angle Incre...

Page 322: ...plays image slices in successive display and clicking it displays image slices by advancing frame by frame Display button Press to start display of n slices of image from the end position to the start position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays ima...

Page 323: ... buttons on the top of the Display panel can be used to vary the speed of successive display of multiple image slices 1 Display the Display panel of the image to be subjected to successive display speed change 2 The buttons as shown below are displayed at the top of the Display panel Usually the Rabbit button is displayed Clicking it displays a list of buttons as shown below Click one of the butto...

Page 324: ...annel or from an image of only one channel To select the target channel s use the Display channel switch buttons Only the channel s being displayed will be used in the animation building Example When only the Ch1 image is displayed only the Ch1 image is used For the switching of channels see section 2 4 3 Switching the Display Channels 3 Click the Arbitrary View option button in the Rendered View ...

Page 325: ... bar shows the progress of building processing During the building the Begin Visualize button turns into the Cancel Visualize button Click the Cancel Visualize button if you want to cancel the building Fig 2 105 Panel Showing the Built Image 12 Click and hold the Display button at the top of the 3D Animation panel The images will rotate so that they can be viewed three dimensionally Click the Stop...

Page 326: ... the following steps 1 Check the Stereo Pair check box 2 Click the Begin Visualize button to start building the images When the building completes the built images are displayed in the 3D Animation panel The status bar shows the progress of building processing During the building the Begin Visualize button turns into the Cancel Visualize button Click the Cancel Visualize button if you want to canc...

Page 327: ...APPLIED OPERATIONS Viewing 3D Image 2 217 Page Fig 2 106 Panel Showing Stereo 3D Images ...

Page 328: ...following steps 1 Check the Stereo Pair check box 2 Click the Begin Visualize button to start building the images When the building completes the built image is displayed in the 3D Animation panel The status bar shows the progress of building processing During the building the Begin Visualize button turns into the Cancel Visualize button Click the Cancel Visualize button if you want to cancel the ...

Page 329: ...APPLIED OPERATIONS Viewing 3D Image 2 219 Page Fig 2 107 Panel Showing 3D Image to be Viewed Through Color Eyeglasses ...

Page 330: ...y The change over time can also be displayed as animation With images composed of multiple slices such as time lapse images the buttons as shown below are displayed at the top of the Display panel 1 Display the Display panel of the image composed of multiple image slices 2 To display XYZT observation images by noticing the progress of time it is required to select the XYT series button under the Z...

Page 331: ...ssive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame Bound button Every time successive display or frame by frame display in a direction ends at the start or end position the display is restarted in the opposite direction Set end position button When successive display or frame by frame display is required se...

Page 332: ...e to end the successive display by using the Display button at the top of the Display panel 6 Click the Set end position button If the end position is not set image No n 1 assuming that the number of images is n becomes the start image automatically 7 Click the Display button The images will be displayed successively from the start position to the end position To stop the successive display click ...

Page 333: ...ay the Enhanced Profile Plot Intensity Map Enhanced Histogram Plot Average Intensity Trace or Integrated Intensity Trace For the operation method see section 2 6 Image Analysis Fig 2 108 Enhanced Profile Plot Window Microsoft Excel is not included in the FLUOVIEW FV300 system Please purchase it separately TIP Save button Saves the profile data in a file using an Excel compatible format Close butto...

Page 334: ...t from FLUOVIEW or display the Start menu by pressing the Ctrl Esc keys 4 Select Programs and issue the Microsoft Excel command 5 From the File menu of Excel select the Open command and open the file saved in step 2 6 When the dialog box as shown below appears click the Delimited option button in the Original Data Type group box then select Windows ANSI from the File Origin drop down list Fig 2 11...

Page 335: ...in the Delimiters group box then select none from the Text Qualifier drop down list Fig 2 111 Dialog Box When the File is Opened by Excel 2 3 8 Click the Next button When the dialog as shown below appears select the General option button in the Column Data Format group box then click the Finish button Fig 2 112 Dialog Box When the File is Opened by Excel 3 3 ...

Page 336: ... images such as Paint The following description takes Paint as example 1 Display the Analyze panel and executes analysis After it display the Enhanced Profile Plot Intensity Map Enhanced Histogram Plot Average Intensity Trace or Integrated Intensity Trace For the operation method see section 2 6 Image Analysis Fig 2 113 Enhanced Profile Plot Window TIP TIP Copy button Copies the plot image to the ...

Page 337: ...ing Image Data to Another Application microVoxel Paint etc To transfer image data to another application the image data should be saved in a file and the file should be transferred 1 Save an image using one of the formats that can be handled by the destination application See sections 2 3 Saving Opening and Shredding Images and 2 3 1 1 Saving Images As a Series for the image saving procedure 2 Exi...

Page 338: ...n below 2 11 1 Writing Characters in Image This facility is used to enter the title acquisition parameters and or notes in an image Some labels are provided in advance Characters can be written either by using these labels or entering desired characters at will 1 Display the Display panel of the image in which you want to write characters 2 Click the Text button in the displayed list of buttons Th...

Page 339: ...tion channel stereo bounds Desired characters can also be entered 1 Click the character in the drop down list of the dialog box 2 Delete the character by pressing the Delete or Back Space key 3 Enter characters from the keyboard When the Set as Default button is pressed after having selected a label it is set as the label displayed permanently at the bottom left of the Display panel 4 Click the Fo...

Page 340: ...ve Overlays dialog box 2 11 2 Displaying the Image Intensity The intensity of any pixel of an image can be displayed without using the Analyze panel 1 Display the Display panel of the image that you want to display the intensity at the front 2 In the list of buttons displayed click the Text button The dialog box as shown below appears Fig 2 116 Active Overlays Dialog Box 3 In the drop down list in...

Page 341: ...on The dialog box as shown below appears Fig 2 117 Active Overlays Dialog Box 3 In the drop down list inside the dialog box select x x hotspot value units to display the X coordinate position or Y hotspot value units to display the Y coordinate position 4 On the image place the mouse pointer on the position that you want to display the X coordinate position or the Y coordinate position and click t...

Page 342: ...rectangle On the image drag the mouse pointer along the diagonal line of the desired rectangle from the top left corner to the bottom right corner To specify a circle or ellipse On the image assume a rectangle circumscribing the circle to be checked and drag the mouse pointer along the diagonal line between opposite corners of the rectangle To specify a polygonal region On the image click the poin...

Page 343: ...g the Comment Size for the operation procedure To display a correct scale it is required that the image has been acquired while the objective magnification setting in the software matches the magnification of the actually used objective A vertical scale can be drawn as well as a horizontal scale 1 Click the mouse on the scale to turn the scale active i e handles displayed around the scale 2 Click ...

Page 344: ... Perform the following operation before starting to draw the scale Click the Text button in the list of buttons 1 When the Annotation dialog box is displayed click its Font button The Font dialog box will appear 2 Change the character font and size using the Font Font Style and Size list boxes 3 Click the OK button to close the Font dialog box 4 Click the OK button to close the Annotation dialog b...

Page 345: ...list of buttons 3 Drag the mouse pointer from the start point to the end point of the desired arrow 4 To change the arrow size see section 2 11 8 Changing the Comment Size The direction indicated by a previously drawn arrow can be changed 1 Click the mouse on the arrow to make the arrow active i e handles displayed on the arrow 2 Click the right button of the mouse 3 Select Edit from the displayed...

Page 346: ... pointer along the desired position in the image 4 To change the color bar size see section 2 11 8 Changing the Comment Size The labels of the color bars can be switched to display or hide 1 Click the mouse on inside the color bars to select The handles appear arround the color bars 2 Right click the mouse 3 Select Edit in the menu to display the sub menu as shown below 4 In the sub menu select Sh...

Page 347: ...ments and click the moue while holding the Shift key to select the second comment and after After making all of the comments to be deleted active i e handled displayed around them click the right button of the mouse on one of the comments and select Delete from the displayed pop up menu 2 11 9 Moving Comment 1 Click the moues on the comment to be moved to make it active i e handles displayed aroun...

Page 348: ...to a cross then dragging the mouse In this case the final positioning of the comment can be determined by placing the mouse pointer outside the areas enclosed by the handles and clicking the left button of the mouse The mouse pointer may be hardly visible depending on the image being displayed In this case use the method of displaying the pop up menu Handle TIP NOTE ...

Page 349: ...pop up menu as shown in Fig 2 118 appears 3 Select Size from the menu 4 The menu pointer turns into When the mouse is moved the comment is magnified or reduced according to the mouse pointer 5 Click the left button of the mouse to determine the size A comment can also be magnified or reduced by selecting it placing the mouse pointer on one of the handles so that the mouse pointer turns into then d...

Page 350: ...og Box 4 Select the desired color from the Color Palette list box To change the comment color automatically set in the FLUOVIEW Setup dialog box For details see the description on the Software panel in section 1 3 1 in MAINTENANCE Overall Setting of FLUOVIEW for detailed operations 2 11 12 Changing the Comment Font 1 Click the mouse on the comment to be changed of font to make the comment active i...

Page 351: ...APPLIED OPERATIONS Entering Comment in Image 2 241 Page Fig 2 120 Properties Dialog Box 4 Set the font type and size using the drop down lists and select the effect ...

Page 352: ...appears Fig 2 121 Print Dialog Box 3 Select the connected printer name from the Name drop down list 4 If it is required to set the detailed data of the printer click the Properties button in the dialog box 5 Click the OK button of the dialog box It is required to install and select the printer driver before the above operation Refer to the Windows manuals for details For the printer operation proc...

Page 353: ...int dialog box can also be displayed by mouse operation on the image 1 Display the image to be output at the printer at the front of the Display panel and click the right button of the mouse on the image 2 A pop up menu as shown below appears 3 Select Print from the menu ...

Page 354: ...y of n slices of image from the start position to the end position sequentially successive display or one by one frame by frame display or to stop successive display Holding the button displays image slices in successive display and clicking it displays image slices by advancing frame by frame Display button Press to start display of n slices of image from the end position to the start position se...

Page 355: ...ying 3 channels may be poor than original 1 Open the two files to be used in creating a new image The image files used in overlay are subjected to the following restrictions The sizes of the images in the two image files should be identical The number of data bits in the two image files should be identical For example it is not possible to overlay a Fluoview Multi Tiff file with a Single TIF S 8 b...

Page 356: ...n below Fig 2 122 Experiments in Memory Dialog Box 3 Click the ADV button in the Experiments in Memory dialog box The Experiment Editor group box appears as shown below Fig 2 123 Experiment Editor Group Box Displayed by ADV Button The frame at the top left of the Experiment Editor group box shows the icon of the image file displayed at the front of the Display panel Experiment List button ADV butt...

Page 357: ...t of the Experiment Editor group box The mouse pointer turns into the image icon during dragging When the image of the second selected image file is composed of multiple image slices the setting of the image slice range is ineffective even when the range is set 6 Up to 3 channels are selected automatically beginning with the first image set in the Experiment Editor group box and connected to Merge...

Page 358: ...he unnecessary channels before selecting required channels 7 Click the Merge button in the Experiment Editor group box A new Display panel showing Merge in the page tab appears and the images including newly merged channel s are displayed in the panel Fig 2 124 Merge Panel Clicking a channel line in this area switches the channel between the selected and deselected status NOTE ...

Page 359: ...d in more than one image file it is possible to use only some of the slices by setting a slice range See section 2 13 1 Setting the Range of Multiple Image Slices for the operation procedure The channel extraction cannot change the image type XYZ XYT etc Therefore it is not possible for example to extract an XY image from an XYZ or XYT image 2 Click the Experiment List button in the toolbar at the...

Page 360: ...periments in Memory dialog box The Experiment Editor group box appears as shown below Fig 2 126 Experiment Editor Group Box Displayed by ADV Button The frame at the top left of the Experiment Editor group box shows the icon of the image file displayed at the front of the Display panel ADV button TIP ...

Page 361: ...hannels of the image set in the Experiment Editor group box are connected to Extract by lines The lines of channels are colored as shown below Ch1 Navy blue Ch2 Light blueCh3 Green Ch4 Yellow green Ch5 Orange Ch6 Red When the mouse pointer is approached to the icon side end of a line connected to Extract the color of the line end turns into yellow Clicking the line in this condition switches the c...

Page 362: ...unnecessary channels as described in the TIP on the previous page so that only the necessary channels are selected 7 Click the Extract button in the Experiment Editor group box A new Display panel showing Extract in the page tab appears and the image of the extracted channel s is displayed in the panel Fig 2 127 Extract Panel ...

Page 363: ...method to be changed 2 14 1 Chart Panel z Series panel This panel is used to change the type title or other settings of the displayed chart as well as to add or delete charts List of displayed charts Move the positioning of the chart selected in the Series Title box Adds the chart selected in the Series Title box by specifying the format Deletes the chart selected in the Series Title box Re titles...

Page 364: ...ws the printed chart or sets the orientation and margins of the print paper Saves the chart in the format of an image file or in the clipboard Sets the margins from the edges of the area in which the chart is displayed When a chart is displayed by zooming or scrolling selects whether the data of only the area contained in the chart axes is to be displayed or not Enables or disables zooming When zo...

Page 365: ...ptimum scale automatically Sets the maximum and minimum values of the chart axes either automatically or manually Sets the number of steps of the values displayed in the chart axis labels Enables or disables the inverted chart display Enables or disables the logarithmic chart display Sets the title of the coordinate axis Sets the angle of displaying the coordinate axis title Sets the angle of disp...

Page 366: ... Auto or None Sets the type color and width of the line along the label Sets the length of the scale line from the coordinate axis to the label Enables or disables the display of scale line in the positions where labels are displayed Sets the type color and width of the scale line on the chart side of the coordinate axis Sets the length of the scale line from the coordinate axis to the chart side ...

Page 367: ...ets the coordinate axis display range with reference to the end point Switches the title display position between the header and footer Enables or disables the title display Switches the title background display range between the entire width of the chart display area and the character area Sets the title character font Sets the type color and width of the title frame Sets the title display positi...

Page 368: ...or Sets the legend character font Sets whether or not the chart is resized according to the legend display Sets the type color and width of the legend frame line Sets the items to be displayed in the legend Usually set Automatic Sets the legend display style This setting is effective when the numerical value display is selected in the Legend Style pull down menu Fine adjusts the positioning of the...

Page 369: ...ves to the next page Moves to the last page Enables or disables the chart axis background display Changes color of Left Bottom and Back wall Background button Sets the background color Border button Sets whether or not the background frame is displayed and sets its color width and type Pattern button Sets whether or not the background pattern is displayed and sets it color width and type Transpare...

Page 370: ...ings in the chart such as label characters together with the chart display Change the zoom ratio Rotates the chart in the horizontal direction with respect to the bottom side Moves the chart display position in the horizontal direction Moves the chart display position in the vertical direction Distorts the chart to change the display effect Sets the 3D chart display effect by varying the angle Rot...

Page 371: ...Point 3D chart is selected Sets the chart color Enables or disables the coloring per coordinate axis Sets the chart pattern Sets the chart color Enables or disables the display varying the color at every point in the chart Sets whether or not chart lines are displayed and sets the color width and type Sets the depth of points in the chart Select the chart series to be set in the Series panel Sets ...

Page 372: ...ints Sets the width of inflection points Sets the height of inflection points Sets the type of inflection points Sets the inflection point background color Resets the background color to the default color Sets whether or not the inflection point borders are displayed and sets the color width and type Enables or disables the chart display inside the legend Sets the shape that the cursor changes whe...

Page 373: ...es or disables the item display in the chart Sets the background color of the item display area Enables or disables the transparent background display Sets the item character font Sets the color width and type of the outer frame of the item display area Sets the color width type and length of the arrows indicating the item display area Sets the contents of the displayed item Enables or disables th...

Page 374: ...ting can be controlled by clicking the right button of the mouse without selecting specific page tabs or buttons zPop up menu of function panel When the right button of mouse is clicked on the page tab of a function panel a pop up menu appears to allow selection of the function panel to be displayed at the front Click the mouse right button here Select the function panel to be displayed at the fro...

Page 375: ...f mouse is clicked on the page tab of a Display panel a pop up menu appears to allow selection of the Display panel to be displayed at the front Fig 2 128 Display Panel Showing Pop up Menu Click the mouse right button here Select the display panel to be displayed at the front from this pop up menu ...

Page 376: ...use is clicked on an comment in image a pop up menu appears to allow editing or deleting the specified comment Fig 2 129 Image Comment with Pop up Menu Moves the comment Resizes the comment Edits the selected arrow or scale Deletes the comment Edits the comment color and font Copies the comment ...

Page 377: ...T setting number of image divisions comment editing Fig 2 130 Display Panel Showing Pop up Menu Displays the image full screen Outputs the image at the printer Saves the image as a series of images Edits the image LUT Edits the image comment Saves the image as a single image Sets the number of image divisions when displaying more than one image Pasts the comment copied on the image Selects all the...

Page 378: ...he File I O Tile or Process panel a pop up menu appears This makes it possible to select the file to be opened when performing tile display inter image operations image channel merger extraction etc Fig 2 131 Function Panel Showing Pop up Menu The pop up menu appears showing the file names that can be selected Select the file name to be processed Click the mouse right button here ...

Page 379: ... Up Table Exit button Exits from FLUOVIEW Annotate button Allows the analysis area to be specified or comment to be drawn in the image Print button Outputs the image at the printer Experiment List button Allows the processing target image to be selected or deselected Full Screen button Displays the image full screen Allows only the image to be displayed by erasing all other image components such a...

Page 380: ...of multiple image slices this button switches between the display according to multiple sections and that according to the progress of time This button is also used to display an extended image Bound Loop buttons Set the repetition direction of the successive display or frame by frame display Magnify reduce button Magnifies or reduces the image Magnification or reduction by 3 1 or 1 3 the original...

Page 381: ...anned not scanned Cttrl 4 Switches the status of the Ch4 check box Ch4 scanned not scanned Cttrl 5 Switches the status of the Ch5 check box Ch5 scanned not scanned PMT voltage gain offset adjustments Key Target Operation Enables and makes variable the LED slider on the left Enables and makes variable the LED slider on the right Decreases the value of an enabled LED slider Fine adjustment Decreases...

Page 382: ...d and area setting Key Target Operation Ctrl Decreases the scan speed Ctrl Increases the scan speed Ctrl Decreases the zoom ratio Ctrl Increases the zoom ratio Z stage setting Key Target Operation Page Up Moves the Z stage up Fine adjustment Shift Page Up Moves the Z stage up Coarse adjustment Page Down Moves the Z stage down Fine adjustment Shift Page Down Moves the Z stage down Coarse adjustment...

Page 383: ...t Operation Ctrl L Switches the transmitted illumination ON OFF Trans Lamp z Image save key Key Target Operation Ctrl S Save the image in the Display panel being displayed as series images SAVE z Panel select keys Main panel Key Target Operation Alt A Selects the Acquire panel Alt F Selects the File I O panel Alt T Selects the Tile panel Alt P Selects the Process panel Alt N Selects the Analysis p...

Page 384: ...e Config sub panel Process sub panels Key Target Operation Alt M Selects the Main sub panel Alt I Selects the Filter sub panel Alt H Selects the Histogram sub panel Alt L Selects the Logical sub panel Analysis sub panels Key Target Operation Alt S Selects the Single sub panel Alt E Selects the Series sub panel Visualize sub panels Key Target Operation Alt O Selects the Orientations sub panel Alt R...

Page 385: ...ton A figure in the shape of button in the window Clicking a command button with the mouse allows the function indicated on the button to be executed Confocal Signifies the possibility of obtaining data on the plane where the irradiated laser beam is focused Contrast Variation change between the brightest and darkest areas in an image Control menu The menu displayed when the control menu box at th...

Page 386: ...the window I Icon An icon is a small figure with characters below it The icon indicates the status in which the window is closed or minimized Iconize This refers to turn a window into an icon display by using the iconize button of the Iconize command in the control menu The application continues to run even after it has been turned into an icon Selecting an icon returns it to the active applicatio...

Page 387: ...ovides a better effect than increasing PMT Voltage above 800 V R Resolution Number of dots composing the image on a screen or printer When the number of dots is increased i e when the resolution is increased the gradation can be displayed in more details Reversed display Display method which indicates that an item or character string is selected and has become the target of the user s next operati...

Page 388: ...e position where the input character is inserted Title bar The horizontal bar at the top of the window that shows the title of the window or dialog box Toolbar The toolbar provides frequently used functions in the form of buttons It can be used any time during execution of any function W Window A large rectangle with a title A window can be opened or closed ...

Page 389: ...tached related module units may also be performed on this screen During setting of the environment data the controls of the hand switch U HS U HSTR and the AX70 microscope frame e g lamp ON OF button will be inoperative Always remember to press the STORE button 2 after setting all the INIT data Fig D 1 If the STORE button is not pressed all the already set data will be erased when another screen M...

Page 390: ...ll be engaged into the light path 3 Rotating the JOG dial select the name of the cube engaged into the light path 4 When END 5 is pressed the first cube name is specified and the previous screen returns 5 To enter all the required cubes repeat the above procedure 6 The currently displayed values can be stored as one set 4 cubes Stored data can then be loaded stored and confirmed for future use 1 T...

Page 391: ...essed the screen shown in FIg D 8 will appear The revolving nosepiece will rotate and the objective at the specified nosepiece position will be engaged 3 Rotating the JOG dial select the designation 3 of the objective engaged into the light path 4 When END 4 is pressed the first objective designation is entered and the previous screen will return 5 to enter the data of other objectives repeat the ...

Page 392: ...Objective PLAPO 40X PLAPO 60XO PLAPO 100XO PLAPO 40XWLSM PLAPO 60XWLSM PLAPO 60XOLSM UPLAPO 10X UPLAPO 20X UPLAPO 20XO UPLAPO 40X UPLAPO 40XOI UPLAPO 60X UPLAPO 60XW UPLAPO 60XWPSF UPLAPO 100XOI UPLFL10X UPLFL20X UPLFL40X UPLFL60XOI UPLFL100XO UPLFL100XOI UMPLFL10XW UMPLFL20XW LUMPLFL40XW LUMPLFL60XW ...

Page 393: ... and the specified turret aperture will be placed at the position of the turret s optical element mounting hole When setting and specifying the turret aperture the optical element is placed at the position of the turret s optical element is placed in the optical axis 3 Using the JOG dial select the designation of the optical element 3 positioned at the turret s optical element mounting hole 4 When...

Page 394: ...ws which DIC prism is suitable for a objective lens Objective Transmitted light DIC prism PLAPO 60X0 ODPAO60 UPLAPO 10X DP10 UPLAPO 20X DPA20 UPLAPO 20X0 DPA20 UPLAPO 40X DPA40 UPLAPO 40XOI DPAO40 UPLAPO 100XOI DP100 UPLFL 10X DP10 UPLFL 20X DP20 UPLFL 40X DP40 UPLFL 100XO DP100 UPLFL 100XOI DP100 ...

Page 395: ...ommunication DIP switch as follows SW1 OFF SW2 ON SW3 ON SW4 OFF SW5 OFF SW6 OFF SW7 ON SW8 ON With the above settings the communication rate is set to 9600 bps data length to 7 bits parity check to None stop bit to 2 bits and delimiter code to CR LF Refer to the instruction manual of your AX microscope for details ...

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Page 397: ... register yourself as a user Log in the system with Administrator Register yourself as a new user 1 From the Start menu select Programs Administrative Tools Common User Manager 2 The User Manager window appears Fig Appendix E 1 User Manager Window 3 Select New User from the User menu 4 The New User dialog box appears Fig Appendix E 2 New User Dialog Box TIP ...

Page 398: ...l Name text box 7 Enter the description on the user in the Description text box 8 Enter a password with up to 14 characters in the Password and Confirm Password text boxes 9 Click the User Must Change Password at Next Login check box to check it The next time you log in with the registered user name you can change the password 10 Click the Group button The Group Memberships dialog box appears Fig ...

Page 399: ...erson who have the authority of Administrator 1 Click the start button at the bottom of the Windows screen 2 When the start menu is displayed select commands Programs Administrative Tools Common Disk Administrator The window as shown below appears Fig Appendix F 1 Disk Administrator Window 3 Select the MO disk drive in the Disk Administrator window The drive to which the MD disk is assigned is var...

Page 400: ... partition of size edit box in the Create Primary Partition dialog box to Maximum size and click the OK button 6 From the Partition menu of the Disk Administrator window select the Commit Change know command 7 When the confirmation message as shown is displayed click the Yes button 8 When the following message is displayed click the OK button 9 In the Disk Administrator window select the MO disk d...

Page 401: ...alog box Then select FAT from the File System drop down list and select FAT and select Default Allocation Unit Size from the Allocation Unit Size drop down list 12 Click the Start button 13 When the confirmation message as shown below is displayed click the OK button 14 When the following message is displayed after completion of formatting click the OK button 15 Click the Close button in the Forma...

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Page 403: ...alog box as shown below appears click the Delimited option button in the Original Data Type group box then select Windows ANSI from the File Origin drop down list Fig Appendix G 1 Dialog Box Displayed When File is Opened with Excel 1 3 4 Click the Next button When the dialog box as shown below appears check the Tab check box in the Delimiters check box then select none from the Text Qualifier drop...

Page 404: ...the Next button When the dialog box as shown below appears click the General option button in the Columns Data Format group box then click the Finish button Fig Appendix G 3 Dialog Box Displayed When File is Opened with Excel 3 3 6 Drag the data to be converted into a chart Sheet ...

Page 405: ...ed The dialog box as shown below appears Fig Appendix G 4 Dialog Box of Chart Wizard 1 5 9 Click the Next button The dialog box as shown below appears Fig Appendix G 5 Dialog Box of Chart Wizard 2 5 10 Select the desired chart type and click the Next button The dialog box as shown below appears Fig Appendix G 6 Dialog Box of Chart Wizard 3 5 ChartWizard button ...

Page 406: ...k the Columns option button under Data Series in 13 Set the data column number that you want to use as the X axis label position in the Use First Column s text box 14 Click the Next button The dialog box as shown below appears Fig Appendix G 8 Dialog Box of Chart Wizard 5 5 15 Enter the chart title in the Chart Title text box 16 Enter the X axis label in the Category X text box and the Y axis labe...

Page 407: ... on the chart black handles will appear around the chart The following operations are available in this condition z Moving the chart Place the mouse pointer on the chart and drag it z Resizing the chart Place the mouse pointer on one of the handles around the chart and drag it To return to the original condition click a place outside the chart To enter reference text use the reference text style T...

Page 408: ...ouble click The dialog box for setting the selected format appears Set the desired format and click the OK button Refer to the Excel manuals for details To return to the original condition click a place outside the chart TIP Chart area format setting Click a margin area inside the chart frame Axis format setting Click a place on a chart axis Plot area format setting Click a place in the plot area ...

Page 409: ...ck the FLUOVIEW Setup icon on the desktop When a dialog box as shown below appears display the Users panel at the front 2 Click the Add a user and Log in button 3 The New FLUOVIEW User dialog box appears FLUOVIEW Setup icon Add a user and Log in button Used to add a new user Reset user to Factory Defaults button Resets the system setups of the users selected in the list box to the factory defaults...

Page 410: ...ing button to close the dialog box The Administrator is the user name which saves the factory defaults of the system setup Appendix H 2 Logging into the FV500 After the user name has been registered and the FV500 started a dialog box for entering the user name appears Enter the user name to log in the FV500 1 Double click the FLUOVIEW icon on the desktop The dialog box for entering the user name a...

Page 411: ...the FLUOVIEW User Login dialog box is not displayed but the system is automatically started for the Administrator Appendix H 3 Deleting a User 1 Double click the FLUOVIEW icon on the desktop The dialog box for entering the user name appears as shown below 2 Enter Administrator in the User Name text box and click the OK button The FLUOVIEW Setup dialog box appears as shown below FLUOVIEW Setup icon...

Page 412: ...be deleted from the list 6 The Delete a user dialog box appears to ask if you really want to delete the user Add a user and Log in button Used to add a new user Reset user to Factory Defaults button Resets the system setups of the users selected in the list box to the factory defaults Delete a user button Deletes the user selected in the list box The Administrator and the user who log in now canno...

Page 413: ... the Yes button if you want to delete the user or the No button if you do not The deleted user name disappears from the list The user is deleted at the moment the OK button is clicked 8 Click the Save New Setup button or Quit w o Saving button to close the dialog box NOTE ...

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Page 415: ...an be changed or a desired folder can be specified directly when loading a saved image Change the default folder only when required There is no need of change if folder C FLUOVIEW IMAGES is all right Folder names are delimited with For example C FLUOVIEW IMAGES means folder IMAGE in folder FLUOVIEW in drive C z When the default folder is C FLUOVIEW IMAGES A different default folder can be set for ...

Page 416: ... I Change of Default Folder for File I O Panel I 2 Page 1 Click the Start button to open the Start menu Then select commands Settings Control Panel System The System Properties dialog box appears as shown below ...

Page 417: ...Select the Environment sub panel 3 Select FLUOVIEWIMAGES under Variable in the User Variables for Administrator list box Administrator of User Variables for Administrator is variable depending on the user name logging in Windows NT User name logging in Windows NT TIP ...

Page 418: ...lder names using backslashes The figure shows an example in which folder IMAGE in drive C is entered 5 Click the Set button Value text box Enter the path name of the default folder Change is reflected OK button Saves the change and closes the dialog box Cancel button Closes the dialog box without saving the change Apply button Saves the change without closing the dialog box ...

Page 419: ...m the next time you start FLUOVIEW z When the default folder is changed to C IMAGES To reset the default folder to the factory setup enter C FLUOVIEW IMAGES in the Value text box Do not change the setup after FLUOVIEWIMAGES in the User Variables for username list box If erroneous setting is made here the system will be unable to be started up New default folder C IMAGES is being open TIP NOTE ...

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Page 421: ...the screen is displayed Syntax x hotspot raw calibrated value units Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied Setup Procedure 1 Enter x as the first character inside 2 Add the following characters to set the display method hotspot Display measurement points with markings raw Display data in pixel values calibrated ...

Page 422: ...e first character inside 2 Add the following characters to set the display method hotspot Display measurement points with markings raw Display data in pixel values calibrated Display data in numerical values µm units Display the unit pixels µm Examples Input character strings Displayed strings represent figures y raw value ĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊ y hotspot raw value ĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊĊ y h...

Page 423: ...ted If raw calibrated is omitted the same setting as when calibrated is specified will be applied 2 T Position With time related images such as XYZ images the T position with respect to the first image can be displayed Syntax t raw calibrated value units Arguments inside can be omitted If raw calibrated is omitted the same setting as when calibrated is specified will be applied 3 Animation With an...

Page 424: ...cedure 1 Enter intensity as the first character inside 2 Add the following characters to set the display method hotspot Display measurement points with markings raw Display data in values between 0 and 4095 calibrated Display data in numerical values µm units Display the unit if this has been set Same as the concentration computation in FV TIEMPO Examples Input character strings Displayed strings ...

Page 425: ...ped the displayed channel numbers are connected by the markings Syntax Channel Appendix J 3 2 Objective Power The objective power used in image capturing can be displayed Syntax Objective Appendix J 3 3 Date of Image Capturing The date at which the image was captured can be displayed Syntax Date Appendix J 3 4 Time of Image Capturing The time of the day at which image was captured can be displayed...

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Page 427: ...This volume describes the user maintenance procedures of the FLUOVIEW FV500 system Please read this volume so that you can use the system for an extended period of time On This Volume ...

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Page 429: ... Configuration 1 6 1 3 1 Overall Setting of FLUOVIEW 1 6 1 3 2 Setting the BX Control Panel 1 14 1 4 Adding the dyeing method 1 20 2 Maintenance of Major System Units 2 1 2 1 Laser Scanning Microscope 2 1 2 2 UV Ar Laser 2 2 2 2 1 Auto Alignment Operation Remote Switch 2 2 2 2 2 Checking Refilling and Replacing Water in Chiller 2 3 ...

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Page 431: ...it To set up the FLUOVIEW software it is necessary that the computer in use already has Microsoft Windows NT Version 4 0 English version installed in its hard disk A vacant space of 100 MB is required for the setup 1 1 Re setup or Updating of the Software Save the current settings and remove the current software before starting re setup or updating with the following procedures 1 Insert the FLUOVI...

Page 432: ...al of the software If the software is removed without saving the current settings the settings in the software program obtained by re setup will be reset to the initial settings 4 Continuously deletion of software is started For deletion carry out according to a wizard 5 Continuously installation of the software of a new version is started Refer to 1 2 New Setup of the Software for details NOTE ...

Page 433: ...W setup CD ROM in the CD ROM drive 2 Select the Start button to display the Start menu then select commands Settings Control Panel Add Remove Programs The Add Remove Programs Properties dialog box as shown below will appear 3 Select the Install button The dialog box as shown below will appear 4 When the Install Program From Floppy Disk or CD ROM dialog box appears select the Next button ...

Page 434: ...allation Program dialog box appears ensure that the CD ROM drive name SETUP EXE is displayed correctly and select the Finish button 6 When the Choose Destination Location dialog box appears confirm the setup destination drive name and directory and select the Next button ...

Page 435: ...Confirm the installation destination directory and select the Next button This will start copying of files 8 When the setup has completed the Setup Complete dialog box appears Select the Yes I want to restart my computer now option button and press the Finish button This will restart the computer ...

Page 436: ...utton to log into FLUOVIEW FV500 Refer to Appendix H 2 Logging into the FV500 for details 3 The dialog as shown below appears for use in saving the system configuration First display the Microscope panel in the front position select the type of microscope and set the extra intermediate attachment magnification to 1 0 Selecting BX51 52 or BX51 52WI and Automatic in the Microscope group box or BX61 ...

Page 437: ...t boxes in the Z Stage sub panel are set according to the value set there For example When 1 0 mm is set as the maximum range for Z series the minimum value for Z starting becomes 500 µm and the maximum value for Z stopping becomes 500 µm Set the backlash of motor operation in the Z Stage sub panel Set the backlash during scanning involving the Z stage Set the range where the stage can move during...

Page 438: ...elect the Delete button To change the details objective name pinhole No magnification of an objective Double click the objective for which you want to change the detailed information When the dialog box appears change the desired information items and select the OK button To add an objective to the list Double click Double Click here at the top of the list on the right When the dialog box appears ...

Page 439: ... to use with FLUOVIEW The setting procedure is similar to the objective setting procedure Objectives panel 7 Display the Coloring Tables panel in the front position In this panel select the characters to be displayed in each of the 8 buttons displayed in the Color Tool dialog box of the FLUOVIEW application and the LUT file to be read out when the button is pressed List of dyes that can be used du...

Page 440: ...o set the numbers of Function and Display panels that can be displayed together Default Image Zoom in Single View group box Select the initial status of display magnification when a single image is to be displayed in a single Display panel Default Image Zoom in Multiple View group box Select the initial status of display magnification when multiple images are to be displayed by displaying multiple...

Page 441: ...p box This data should usually be left in the default values and does not have to be changed Delay shutter changes sec text box Enter the opening closing wait time of the shutter in the TD unit in seconds Delay for bulb changes sec text box Enter the ON OFF wait time of the lamp in the TD unit in seconds Automatic Black level adjustment group box Select Enabled Disabled to enable disable the autom...

Page 442: ... each channel And corresponds to the order of the scanning unit from the top to bottom Beam Splitter Sets the beam splitter of each channel From the left this shows the beam splinter of Ch1 Ch2 and Ch3 And corresponds to the order of the scanning unit from the top to bottom Barrier Filter Sets the barrier filter of each channel And corresponds to the order of the scanning unit from the top to bott...

Page 443: ... 12 The Registration panel have been set at the factory and do not need to be changed here 13 After completing the setup select the Save New Setup button on the bottom left of the panel Selecting Quit w o Saving exits the panel without saving the system setup ...

Page 444: ...he BX Control Panel window Mirror Unit group box Sets up the name and color of the cube turret Nosepiece group box Sets up the name color of the objective and sets the magnification N A refractive index n number of the confocal pinhole and the condenser worked with to each objective Condenser Turret group box Sets up the name and color of the universal condenser Filter Turret group box Sets up the...

Page 445: ...e Editing in the Nosepiece group box Setting the Name Setting the Objective Magnification Setting the condenser worked with Setting the magnification of the objective Setting the N A of the objective Setting the number of the confocal pinhole Setting the refractive Setting the color of the button disengaged from the light path Section 1 3 1 3 1 Setting the color of the button engaged into the ligh...

Page 446: ...disables the turret itself 1 3 2 2 Setting the Name 1 Editing the name of the buttons in the Mirror Unit Condenser Turret and Filter Turret group boxes 1 In the Mirror Unit Condenser Turret or Filter Turret group box right click the mouse on the button whose name is to be edited The pop up menu as shown below appears 2 Select Edit The Edit dialog box as shown below appears 3 Enter a name into the ...

Page 447: ... whose color is to be changed The pop up menu as shown below appears 2 Select Color The Color dialog box as shown below appears 3 Select the color which you want to set in the color palette and click the OK button Name of the Objective drop down list Select the name of the objective in the list or enter the name with keyboard Name of the Linked Condenser drop down list Select the condenser worked ...

Page 448: ...olor of the button of the cube the objective and the condenser engaged into the light path Right click the mouse on the area outside of the buttons in the Mirror Unit Nosepiece Condenser Turret or Filter Turret group box 2 Select Selected Hole Color The Color dialog box as shown below appears 3 Select the color which you want to set in the color palette and click the OK button ...

Page 449: ...Or click the Quit w o Saving button to cancel the setup and close the window Then the dialog box as shown below appears If you want to save click the Yes button if you do not want to save click the No button After saving is selected in the Microscope Setting window the original setting is not returned even if the Quit w o Saving is clicked in the FLUOVIEW Setup dialog box Save New Setup button Qui...

Page 450: ...aser type excitation wavelength and emission wavelength Add the dyeing method using the Fluorescence panel in the Fluoview Setup dialog box 1 Double click the FLUOVIEW Setup icon on the desktop The FLUOVIEW User Login dialog box as shown below appears 2 Enter user name into the User Name text box and click the OK button to log into FLUOVIEW FV500 When using the system for one user the FLUOVIEW Use...

Page 451: ...HeCd As the laser to use helium cadmium laser should be set in advance When HeCd is not displayed check the HeCd check box in the Laser Equipment panel Double click here to add the dyeing method Name of the Dye text box Enter the name of the dyeing method Laser Type drop down list Select the laser to use Emission Wave Length group box Enter the emission wavelength In case of the 1 wavelength and 2...

Page 452: ...th group box e g 442 8 Enter the value of the emission wavelength into the WaveLength 1 text box in the Emission Wave Length group box e g 480 photometry enter the value of the wavelength into the WaveLength 2 text box too 9 Click the OK button to close the Edit Dye dialog box 10 Select the Save New Setup button to close the FLUOVIEW Setup dialog box TIP ...

Page 453: ...tted as part of user maintenance activities Such attachment and removal should be performed as part of service activities For detailed imformation refer to our service representatives 1 Transmitted lamp housing 2 Transmitted light fiber cable 3 Cube turret of reflected light fluorescence unit 4 Objective revolving nosepiece 5 Objectives 6 Condenser 7 Differential interference slider 8 Excitation f...

Page 454: ...izes the laser oscillation by adjusting the unstability in output due to mirror alignment error resulting from long period of use It is recommended to perform auto alignment about once a month After warming up the laser head for more than 30 minutes press the remote switch connected to the rear of the laser head once The auto alignment starts and completes in some tens of seconds The indicator on ...

Page 455: ...d change the distilled water The check period is about once every six months Note that the water tends to be polluted when the weather is warm or when the heat exchanger has been left unused for a long period 1 Switch off the main circuit breaker of the heat exchanger and unplug its power cable from the power outlet 2 Remove the 4 top cover retaining screws and remove the top cover Front panel Top...

Page 456: ...Maintenance of Major System Units UV Ar Laser 2 4 Page 3 Remove the water tank cover and check the level and pollution of water in it Specified level Water tank Distilled water ...

Page 457: ...which comes inside the tank 4 Refill new distilled water until the specified level Run the heat exchanger manually and check the water level again If it is below the specified level refill additional distilled water 5 Close the water tank with the lid place the top cover and tighten the 4 retaining screws tightly using a screwdriver 6 Plug the power cord into the power outlet and set the main circ...

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Page 459: ...es the treatment against possible troubles In case of trouble please read volume before calling for service If the normal operation cannot still be restored please contact your local Olympus representative On This Volume ...

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Page 461: ... displayed 1 4 13 The scale of the Z Stage sub panel in the Acquire panel cannot be moved 1 4 14 The fine movement knob of microscope cannot be turned or is not smooth 1 4 15 The image cannot be saved in the disk 1 4 16 The image cannot be output at the printer 1 4 17 The FLUOVIEW software cannot be started 1 4 18 The mouse pointer in the screen cannot be moved by moving the mouse 1 5 19 The power...

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Page 463: ... scanning sets the LSM light path automatically Set the light path selector dial to the LSM light path 1 1 1 1 2 4 OPERATION INSTRUCTIONS An excitation tube or analyzer for visual observation is engaged in the microscope When the AX70A is used starting scanning disengages the excitation cube from the light path automatically Disengage the excitation tube and analyzer from the light path 1 1 1 1 2 ...

Page 464: ...UCTIONS The photomultiplier tube of the channel set with the detection mode setting slider is not set Check the Ch1 Ch2 Ch3 Ch4 or Ch5 check box in the Acquire panel 1 2 8 3 2 2 1 4 3 OPERATION INSTRUCTIONS 2 Transmitted image cannot be observed The transmittance detection channel is not selected In the Acquire panel check the check box of the Ch with which Transmitted is displayed 2 2 3 3 OPERATI...

Page 465: ...image The excitation wavelength does not match the barrier filter type or the barrier filter is not used Select suitable barrier filter for the excitation length 1 2 6 1 3 2 4 OPERATION INSTRUCTIONS 9 Image is blurred or out of focus The focusing is not correct Adjust focusing with visual observation Instruction manual of microscope 10 Image is dark and noisy The fluorescent dye is pale Apply opti...

Page 466: ...ruction manual of MO disk The MO disk is not recognized Check the cable connection Instruction manual of MO disk The disk is not loaded in the drive Insert the disk properly Instruction manual of MO disk The disk is write protected Release the write protection Instruction manual of MO disk The disk is not formatted either floppy disk or MO disk Format the disk before use Appendix F OPERATION INSTR...

Page 467: ...ce then replace the huse 20 The current limit indicator on the UV Ar laser power supply is blinking The laser is in standby condition Start the FLUOVIEW software then click the On button for UV Ar Laser in the Laser Unit group box in the Optical System Configuration window to restart laser oscillation 1 3 2 4 OPERATION INSTRUCTIONS 21 The error indicator on the UV Ar laser power supply will not tu...

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Page 470: ...r Drive Melville NY 11747 3157 U S A OLYMPUSSINGAPOREPTELTD 491B River Valley Road 12 01 04 Valley Point Office Tower Singapore 248373 OLYMPUSOPTICALCO U K LTD 2 8 Honduras Street London EC1Y OTX United Kingdom OLYMPUSAUSTRALIAPTY LTD 104 Ferntree Gully Road Oakleigh Victoria 3166 Australia This publication is printed on recycled paper Printed in Japan 2001 05 1 6 ...

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