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INTRODUCTION TO LASER SCANNING MICROSCOPY

LSM 510

Contents

B 40-051 e 07/98

3-1

CHAPTER 3

INTRODUCTION TO
LASER SCANNING MICROSCOPY

CONTENTS

INTRODUCTION TO LASER SCANNING MICROSCOPY .............................................3-3

3.1

Principle of Laser Scanning Microscopy ........................................................................3-3

3.2

Three-Dimensional Presentations of LSM Image Stacks .................................................3-4

3.3

Optical Diagram of the LSM 510 (Schematic) ...............................................................3-6

3.4

Performance Features of the LSM 510 .........................................................................3-7

3.4.1

Optical and mechanical aspects ...................................................................................3-7

3.4.2

Microscope equipment of the LSM 510 system ............................................................3-8

3.4.3

Computer hardware and software............................................................................. 3-11

Содержание LSM 510 Inverted

Страница 1: ...I LSM 510 Laser Scanning Microscope Operating Manual...

Страница 2: ...anual is not covered by an update service Unless expressly authorized forwarding and duplication of this document and the utilization and communication of its contents are not permitted Violations wil...

Страница 3: ...ODUCTION LSM 510 B 40 051 e 07 98 III Developed in Collaboration with the European Molecular Biology Laboratory EMBL PF 102209 Meyerhofstr 1 D 69012 Heidelberg Phon 49 0 62221 387 0 Fax 49 0 62221 387...

Страница 4: ...INTRODUCTION LSM 510 IV B 40 051 e 07 98...

Страница 5: ...to find out which settings and methods are capable of improving or accelerating a procedure The HOT SURFACE symbol warns against hazards for the user that might arise when touching the lamp housing du...

Страница 6: ...1 Configuration 7 000000 1027 082 Configuration 8 000000 1027 083 Configuration 9 000000 1027 084 Configuration 10 000000 1027 085 Configuration 11 000000 1027 086 Configuration 12 000000 1027 087 Con...

Страница 7: ...0000 1013 187 XY scanning stage for Axiovert 100 M BP 000000 1017 917 XY scanning stage for Axioplan 2 MOT 000000 1027 823 Filter changer 18 mm 453070 0000 000 Set of INDO filters 447960 0000 000 Set...

Страница 8: ...section also outlines the ways to present LSM image series in three dimensions and introduces you to the performance features of your LSM 510 4 In the Operation section you will find the most importa...

Страница 9: ...INTRODUCTION LSM 510 B 40 051 e 07 98 IX 1 Notes on Device Safety 2 LSM 510 Setup Requirements 3 Introduction to Laser Scanning Microscopy 4 Operation 5 Software 3D Description...

Страница 10: ...INTRODUCTION LSM 510 X B 40 051 e 07 98...

Страница 11: ...1 NOTES ON DEVICE SAFETY 1 3 1 1 General 1 3 1 2 Regulations 1 3 1 3 Notes on setting up the microscope system 1 4 1 4 Notes on handling the computer and data media 1 5 1 5 Notes on care maintenance...

Страница 12: ...NOTES ON DEVICE SAFETY Contents LSM 510 1 2 B 40 051 e 07 98...

Страница 13: ...ions belonging to the system conscientiously before operating the LSM 510 You can obtain additional information on the hardware configuration delivered and on optional system extensions from the manuf...

Страница 14: ...of extension leads Before connecting the mains cables please check whether your mains voltage corresponds to the voltage specified on the rating plate of the laser module Maintenance repairs modificat...

Страница 15: ...kettes avoid data losses by protecting them against extreme temperatures moisture and magnetic fields The data on a diskette is stored in the form of magnetic signals To some extent monitors telephone...

Страница 16: ...rsons authorized and trained for this purpose by the manufacturer Damaged units or parts may only be repaired or maintained by the responsible service agency Care operations that may be carried out by...

Страница 17: ...ngerous laser radiation area is limited to the beam path and to a distance of up to around 10 cm from the specimen Nevertheless you should observe the following warnings If necessary insofar as specif...

Страница 18: ...ou will receive a free replacement The label means Do not remove securing screw as otherwise laser beam will escape For use by service only Fig 1 1 Warning and information labels on the Axiovert 100 M...

Страница 19: ...NOTES ON DEVICE SAFETY LSM 510 Warning and information labels B 40 051 e 07 98 1 9 Fig 1 2 Warning and information labels on the Axioplan 2 microscope with LSM 510 scanning module...

Страница 20: ...ER FAC E CO N 2 AR GO N LA SE R PO WE R SU PP LY LG N 78 12 0 l Lase r Ferti gung Gmb H Omnichrome IO N LA SE R PO WE R SU PP LY LASER RADIATION AVOID DIRECT EXPOSURE TO BEAM ARGON LASER 450 514 200mW...

Страница 21: ...RADIATION IS EMITTED FROM THIS APERTURE Laser Fertigung GmbH LGK 7774 TATZENPROMENADE 1a D 07745 JENA MADE IN GERMANY Q4001 K7784 S NO 000 CERTIFICATION THIS PRODUCT CONFORMS TO ALL APPLICABLE STANDAR...

Страница 22: ...NOTES ON DEVICE SAFETY Warning and information labels LSM 510 1 12 B 40 051 e 07 98...

Страница 23: ...LSM Power 3 2 6 2 2 3 Separate connection 2 6 2 3 Physical Dimensions 2 6 2 4 Dimension of shipment crates 2 7 2 5 Environmental Requirements 2 7 2 6 Vibrations 2 7 2 7 Laser Specifications 2 8 2 7 1...

Страница 24: ...LSM 510 SETUP REQUIREMENTS Contents LSM 510 2 2 B 40 051 e 07 98...

Страница 25: ...EMENTS 2 1 Space Requirements 2 1 1 LSM one microscope large system table 320 cm x 220 cm Fig 2 1 2 1 2 LSM with Ar laser UV 340 x 260 cm We recommend placing the cooling unit of the Ar laser UV in a...

Страница 26: ...SM 510 2 4 B 40 051 e 07 98 2 1 3 LSM with Ar laser UV and two microscopes 450 x 220 cm We recommend placing the cooling unit of the Ar laser UV in a separate room to prevent heat accumulation and vib...

Страница 27: ...25 A Phase 1 1 8 kVA max Phase 2 2 kVA max Power consumption 2000 VA per phase 2000 VA per phase Power plug CEE red 230 V 16 A 3 phases N PE phases 1 and 2 connected CEE yellow 115 V 32 A 3 phases N P...

Страница 28: ...laser 2 kW 2 2 3 Separate connection Ar laser UV 7 kW 2 3 Physical Dimensions Length cm Width cm Height cm Weight kg Large system table 150 80 78 100 Small system table 65 80 78 60 Scanning Module 25...

Страница 29: ...ter 120 80 90 80 UV laser unit 125 55 50 100 UV cooling unit 120 60 90 50 2 5 Environmental Requirements Operation specified performance T 22 C 3 C Operation reduced performance T 10 C to 35 C Storage...

Страница 30: ...wer consumption 7400 VA With heat exchanger LP5 Water flow 8 0 l min max 16 l min Water pressure 1 4 4 2 kg cm Water temperature 10 60 C at 8 0 l min Power to water cycle max 4500 W Power from power s...

Страница 31: ...100 240 V Line frequency 50 60 Hz Max current 1 phases at 25 A Power consumption 2000 VA Cooling fan on top of laser head 2 7 5 Omnichrome 50YB 643 171B 488 568 nm 30 mW laser power class 3 B Line vo...

Страница 32: ...ividually driven galvanometric scanners Scanning speed Up to 2 6 frames sec 512 x 512 pixels Field resolution Max 2048 x 2048 pixels individually adjustable for each axis Field of view 10 x 10 mm with...

Страница 33: ...er 488 nm 15 mW ArKr laser 488 568 nm 30 mW Fuses and automatic circuit breakers for 230 V G type fuse 5 x 20 mm slow blow 3 15 A H 250 V acc to IEC 127 2 circuit breakers C 10 A for 110 V G type fuse...

Страница 34: ...LSM 510 SETUP REQUIREMENTS Laser Module VIS Laser Module UV LSM 510 2 12 B 40 051 e 07 98...

Страница 35: ...ER SCANNING MICROSCOPY 3 3 3 1 Principle of Laser Scanning Microscopy 3 3 3 2 Three Dimensional Presentations of LSM Image Stacks 3 4 3 3 Optical Diagram of the LSM 510 Schematic 3 6 3 4 Performance F...

Страница 36: ...INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 3 2 B 40 051 e 07 98...

Страница 37: ...scent specimens would disturb the in focus image of object point of interest resulting in a blurred image of poor contrast The problem is therefore how to capture only the light coming immediately fro...

Страница 38: ...information is a gallery showing the individual slice images sections of a stack arranged side by side with each slice apart from the next by a defined selectable interval on the Z axis Virtually inf...

Страница 39: ...s Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates i e X Y Z angle of rotation and angle of tilt Topography forthcoming A comput...

Страница 40: ...men through an objective in a diffraction limited mode Light emitted at the focal plane and at planes below and above it is directed via an XY scanner onto a main dichroic beam splitter MDBS which sep...

Страница 41: ...s image acquisition channels usable for reflection or fluorescence and an additional transmitted light channel are ideal for the investigation of multiple fluorescence specimens Separately in each of...

Страница 42: ...H DIC is firmly fixed to the stand where no operating elements can be found for the nosepiece Operation will be done LSM 5 software controlled The Restriction of revolver hight to protect the objectiv...

Страница 43: ...TV adapters 452982 83 92 94 95 97 98 The light reaches the ports over full mirrors Therefore all light is available at side port base port or for conventional microscopy Bringing the beam path to the...

Страница 44: ...e is mounted a LSM 5 software controlled switching mirror fully motorized Alternatively the light is directed to the LSM 5 T light detector or enables conventional transmitted light observation d The...

Страница 45: ...le operator interface level a result will be achieved after a few prompts graphical prompting of the user in conjunction with automatic setting of many parameters is an ideal tool for daily routine jo...

Страница 46: ...INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 3 12 B 40 051 e 07 98...

Страница 47: ...ER SCANNING MICROSCOPY 3 3 3 1 Principle of Laser Scanning Microscopy 3 3 3 2 Three Dimensional Presentations of LSM Image Stacks 3 4 3 3 Optical Diagram of the LSM 510 Schematic 3 6 3 4 Performance F...

Страница 48: ...INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 3 2 B 40 051 e 07 98...

Страница 49: ...scent specimens would disturb the in focus image of object point of interest resulting in a blurred image of poor contrast The problem is therefore how to capture only the light coming immediately fro...

Страница 50: ...information is a gallery showing the individual slice images sections of a stack arranged side by side with each slice apart from the next by a defined selectable interval on the Z axis Virtually inf...

Страница 51: ...s Oblique sections A section through the stack is made along an oblique plane defined by the selection of five coordinates i e X Y Z angle of rotation and angle of tilt Topography forthcoming A comput...

Страница 52: ...men through an objective in a diffraction limited mode Light emitted at the focal plane and at planes below and above it is directed via an XY scanner onto a main dichroic beam splitter MDBS which sep...

Страница 53: ...s image acquisition channels usable for reflection or fluorescence and an additional transmitted light channel are ideal for the investigation of multiple fluorescence specimens Separately in each of...

Страница 54: ...H DIC is firmly fixed to the stand where no operating elements can be found for the nosepiece Operation will be done LSM 5 software controlled The Restriction of revolver hight to protect the objectiv...

Страница 55: ...TV adapters 452982 83 92 94 95 97 98 The light reaches the ports over full mirrors Therefore all light is available at side port base port or for conventional microscopy Bringing the beam path to the...

Страница 56: ...e is mounted a LSM 5 software controlled switching mirror fully motorized Alternatively the light is directed to the LSM 5 T light detector or enables conventional transmitted light observation d The...

Страница 57: ...le operator interface level a result will be achieved after a few prompts graphical prompting of the user in conjunction with automatic setting of many parameters is an ideal tool for daily routine jo...

Страница 58: ...INTRODUCTION TO LASER SCANNING MICROSCOPY Performance Features of the LSM 510 LSM 510 3 12 B 40 051 e 07 98...

Страница 59: ...Z Sectioning 4 19 4 4 Overview of the Menu Items 4 22 4 5 Image Acquisition Acquire 4 27 4 5 1 Laser settings 4 28 4 5 2 Microscope settings conventional microscopy 4 30 4 5 3 Beam path Configuration...

Страница 60: ...ge 4 151 4 10 2 Macro Control 4 151 4 10 3 Recording and running of macros 4 153 4 10 4 Assignment of macros to the macro buttons in the main window 4 154 4 10 5 Editing and debugging of macros 4 155...

Страница 61: ...the microscope the scanning and laser modules tools objectives filters CLM 32 and the image acquisition process and presents and analyzes the image data It is based on the network capable graphic 32 b...

Страница 62: ...itch If this switch is not used turn the system on with the I button on the laser module in addition the jumper plug supplied must be plugged at the POWER REMOTE CONTROL terminal Turn the REMOTE CONTR...

Страница 63: ...on the monitor Fig 4 2 4 2 2 Log on to WINDOWS NT Press the three keys Ctrl Alt and Del at the same time The Logon Information dialog box appears on the monitor permitting you to log on to operating...

Страница 64: ...510 dummy A program that starts the LSM in only dummy mode Useful in case of working with stored images with a separate workstation or when demonstrating the system without microscope scan module ele...

Страница 65: ...p double click on Start LSM 510 icon The LSM 510 Switchboard menu appears on the screen 2 From the LSM 510 Switchboard menu click on the Scan New Images button and Start Expert Mode button The LSM wil...

Страница 66: ...click on the Open button instead of New button 3 Type in a data base name in the File name field The name can consist of as many characters as you like Before clicking the create button in the Create...

Страница 67: ...k on the laser s which has the appropriate wavelength to excite the dyes labeling your specimen 4 In the case of the argon and UV laser click on the Standby button first Warming Up appears and when th...

Страница 68: ...8 4 3 4 Look in the Microscope and Visually Set Up Your Specimen 1 Click on the Micro and VIS buttons from the LSM 510 tool bar The Axioplan Control menu or the Axiovert Control menu appears on the sc...

Страница 69: ...e Axioplan Control menu or Axiovert Control menu Put specimen on the stage make sure the specimen is mounted securely and flat 4 You can view the specimen in either fluorescence reflected light or tra...

Страница 70: ...d the area of interest is selected close the menu and click on the LSM button from the LSM 510 tool bar The software will direct you regarding the appropriate setting of the silver slider If required...

Страница 71: ...ng Configurations window appears on the screen 2 A list of configurations will appear by clicking on the button Choose a configuration from the list based on what you need to image from your specimen...

Страница 72: ...510 4 14 B 40 051 e 07 98 4 3 6 Laser Scanning 1 Click on the Scan button from the LSM 510 tool bar The Scan Control window appears on the screen 2 Click on the Find button on the right side of the S...

Страница 73: ...right side of the image 5 If you want to optimize the intensity of an image you can adjust the detector gain sensitivity and Amplifier Offset black level as follows 6 Click on the Channels button from...

Страница 74: ...left and right arrows to make the adjustment instead of dragging the slider bar 10 To adjust the black level background use Ampl Offset 11 Also try adjusting the microscope by manual focusing Sometim...

Страница 75: ...interest by scanning the laser into a smaller area of the field of view This function can actually increase the X Y resolution of an image 15 A red box will appear on the image Press and hold the mou...

Страница 76: ...your image The setting average of 8 Number 8 should improve signal noise dramatically however the image acquisition rate will be slower 18 If your specimen is sensitive to photobleaching you can atte...

Страница 77: ...n 1 Click on the Z Stack button on the Scan Control menu 2 If you have reduced the scan speed or have set image averaging you should use the fast scanning mode to find the lowest and highest points of...

Страница 78: ...down manually clockwise until the image of the specimen begins to disappear then click on the Mark First button in the Mark First Last register 7 Now move the focus in the opposite direction up until...

Страница 79: ...he air table or the microscope until z sectioning is completed Each successive z slice can be viewed by changing to the Gallery Mode This button is located on the right side of the image 11 A black ba...

Страница 80: ...an New Images Clicking on this button activates the complete LSM hardware Use Existing Images This item allows you to process and analyze previously acquired images with the LSM software In this mode...

Страница 81: ...You need to set all parameters and functions upon your own decision this mode therefore provides you with the greatest flexibility of operation It is also possible however to call up stored configura...

Страница 82: ...e shown in the second bottom toolbar The buttons of the main menu upper toolbar have the following meanings File Open save import and export of image data Acquire Calling up and setting the necessary...

Страница 83: ...acquired images Shift allows you to perform a pixel shift correction Fig 4 25 3D For three dimensional image processing Fig 4 26 Macros Makes it possible for the user to store frequently used processe...

Страница 84: ...Under this menu item access to the colouring table will be enabled In the Settings for User window you can specify essential operating modes and informative help organized by registers which have an...

Страница 85: ...s to be selected for the scan procedure The EditBleach function is used to bleach a defined freely selectable area within the scanning field Upon selecting Stage you can set the focus Z coordinate and...

Страница 86: ...rise and Ar multiline Laser Click on the Standby button Wait for the laser to heat up until the Status ready Standby message appears Click on the On button Status ready On appears Use the Power slider...

Страница 87: ...0 software If the LSM 510 software is already running and you want to use the UV laser do the following 1 Close the LSM 510 software 2 Switch on the power supply of the Enterprise power potentiometer...

Страница 88: ...tive to suit the job dry water or oil immersion see inscriptions on objectives w for water and oil for immersion oil Click on the Micro button This opens the Microscope Control window on the screen If...

Страница 89: ...e setting light intensity of the last session which was not remote controlled is restored on exit of the program In the Objectives list box select the required objective as follows Open the list box C...

Страница 90: ...se an objective which is not contained in the nosepiece proceed as follows 1 Change objectives 2 Click on the Change Objectives icon The submenu Modify Objectives Settings appears Fig 4 34 3 Choose th...

Страница 91: ...ire B 40 051 e 07 98 4 33 4 Click on the Maintain button in the main menu and then on the Reboot button in the submenu 5 Confirm the selection in the Reboot Components dialog box by clicking on the Re...

Страница 92: ...l go This opens the light path for specimen observation through the eyepieces Actuate the shutter switch 4 37 4 to open the light path of the halogen lamp and control its brightness with the potentiom...

Страница 93: ...s in transmitted light position To avoid excessive bleaching expose the specimen to the minimum possible irradiation i e keep the irradiation time as short as possible In the Reflector Turret list box...

Страница 94: ...quest DAPI BP 365 FSET01 FT 395 LP 397 TRITC BP 546 FSET15 FT 580 LP 590 Use the focusing drive 4 37 5 to focus the required object plane Select specimen detail by moving the stage in X and Y via the...

Страница 95: ...s far as it will go This opens the light path for specimen observation through the eyepieces Push in the sideport slider 4 39 2 as far as it will go Pull out the VIS slider 4 39 3 as far as it will go...

Страница 96: ...far as possible This opens the light path for specimen observation through the eyepieces Push in the sideport slider 4 39 2 as far as possible Pull out the VIS slider 4 39 3 as far as possible In the...

Страница 97: ...the Config button This opens the Configuration Control window in which you can configure the system for scanning The window contains two panels Beam Path and Channel Assignment and Ratio Settings Fig...

Страница 98: ...ctory preset or created by the user on the Store Apply Configuration panel Saving a user created configuration under a freely selectable name Deletion of a configuration A configuration stored in the...

Страница 99: ...figuration The selected configuration is shown in the Configurations status box Click on the Apply button This results in the stored instrument parameters being taken over for active use The optical d...

Страница 100: ...four emission ranges it is possible to use eight channels or the maximum of four tracks in one scanning procedure If a second track or further tracks are used the scan parameters can be changed as re...

Страница 101: ...re B 40 051 e 07 98 4 43 2 Click on the Show Tracks button on the right side of the window Within the Configuration Control window the additional List of Tracks panel appears The following functions a...

Страница 102: ...ine the configuration of the track in the Beam Path and Channel Assignment panel or select an existing one from Track Configurations and activate it via the Apply button 3 Store the name of a configur...

Страница 103: ...e parameters are reset Fast Switch button The scanning procedure can be made faster Only the Acousto Optical Tunable Filters AOTF are switched and no other hardware components Line button The new para...

Страница 104: ...e Acquisition Acquire LSM 510 4 46 B 40 51 e 07 98 Clicking the Scan button in the LSM 510 tool bar and the Channels button in the Scan Control window allows viewing of the channel configuration set b...

Страница 105: ...uisition Acquire B 40 051 e 07 98 4 47 The following scan image shows the result with two defined tracks plus the ratio track and the overlay Fig 4 47 2 Track Ch 1 2 Ch 4 2 Overlay 1 Track Ch 1 1 Ch 3...

Страница 106: ...trol window will display the defined channels and the ratio channel in the Channel Settings panel Clicking on the R1 1 button for the ratio channel will open the following window Three formulas are av...

Страница 107: ...f you deactivate Line On the laser wavelengths for Enterprise and argon lasers are deselected by means of the AOTF i e these lasers change into standby status If you interrupt your work with the LSM f...

Страница 108: ...very sensitively to the job Click on Line On This activates the selected laser power for use This is indicated by the laser status line displaying On By clicking on the Excitation button you can chec...

Страница 109: ...To select a beam splitter click on the respective line of the list The selected beam splitter moves into the beam path Photomultipliers On the Beam Path and Channel Assignment panel move the cursor on...

Страница 110: ...hannel Colors window Via a reticule in the Define Color dialog box any desired color can be produced Clicking on the Add button allows the color to be used for further channel coloring Choose the desi...

Страница 111: ...into the beam path in front of the PMT Depending on the application it may be necessary to insert additional mirrors dichroic beam splitters or neutral glass filters between the main dichroic splitter...

Страница 112: ...and 4 5 2 2 The specimen must have been positioned and focused by conventional eyepiece observation in fluorescent or transmitted light Axiovert 100 M BP Perform the settings in the same way as descri...

Страница 113: ...Use the Scan Speed slider to set the scanning speed needed Recommended 7 for the first scan Speed 10 fast xy only for fast image acquisition during parameter setup Pixel time and scan time will be sh...

Страница 114: ...ick on Channels in the upper toolbar This opens the new dialog boxes Channel Settings and Excitation of Track showing the previously set parameters which can however be edited Optimize the image quali...

Страница 115: ...he Z Sectioning register Use the Num Slices slider to select the number of slices in the stack Use the Interval m slider to select the Z interval between successive slices Use the Current Slice slider...

Страница 116: ...al depending on the objective used and the pinhole diameter setting Red and other colors Presentation of the actual data set by the operator helps to optimize stack creation Click on the XY scan butto...

Страница 117: ...Range For a useful evaluation of the z stack you must proceed as follows Click on the Cutline button The image window shows the course of the red cutline Clicking on the cutline allows you to remove t...

Страница 118: ...e button The image window marks the range to be scanned by red outlines Not only the current slice green cutline but also the red range lines can be removed by moving the cursor in the image window Fi...

Страница 119: ...and press Mark Last button The XY Cont Scan must be finished via Stop before acquisition of Z Stacks can be startet via Start The First Mid and Last buttons allow a fast overview of the first middle a...

Страница 120: ...tivated objective can be changed number of pixel and scan speed can be set desired average and number of gray shade can be chosen as well as uni or bidirectional scan and the desired zoom rotation and...

Страница 121: ...area symbols are available in the form of buttons 3 Click on the marking area and keep the mouse button pressed to drag the area into the region of interest in the scanning image The marking area wil...

Страница 122: ...ing image Auto button A defined color from the list of colors can be assigned to the ROIs In the Auto position the outlines of the dragged ROIs are automatically colored differently button This button...

Страница 123: ...Acquire B 40 051 e 07 98 4 65 The following is an example of a scanning procedure where the ROI function was activated Only the regions of interest defined before are visible in the scanning image th...

Страница 124: ...n 2 Set the relevant parameters for time control in the Start Series End Series and Cycle Delay boxes 3 Use the Set 1 to Set 7 buttons to set markers during the scanning procedure which will allow you...

Страница 125: ...OPERATION LSM 510 Image Acquisition Acquire B 40 051 e 07 98 4 67 Time Series Control window enabling the entry of parameters for time series control Fig 4 67...

Страница 126: ...ies function Both the time and the markers set during the scanning procedure are projected in the image series in different colors If the cursor is moved to a marker position in the scanning image the...

Страница 127: ...ed as follows Click on the Edit Bleach button The Bleach Control window appears The Start Settings panel allows you to determine when an how the bleaching process shall be triggered only works in conn...

Страница 128: ...ity under Excitation of Bleach Track in the Bleach Control window The bleaching process will be started via the Bleach button However it is also possible to start the bleaching process via Bleach butt...

Страница 129: ...ds which were already created and optimized in the Expert Mode If User Defined Examination Methods are also to be used in the Routine Mode they must be exported in the Routine Mode first Apart from Us...

Страница 130: ...click on the Standard button An icon appears beside the name Click on the Close button in the Select Method To Add window The standard examination methods are taken over and are then available in the...

Страница 131: ...in the same way as in the Expert Mode Click on the Next button The Routine Mode Image Setup window will appear Click on Single Scan or Cont Scan to trigger the scanning procedure If required optimize...

Страница 132: ...OPERATION Routine Mode LSM 510 4 74 B 40 051 e 07 98 Click on Save or Save As to store the acquired image Click on Finish to exit the Routine Mode and return to the LSM 510 switchboard menu Fig 4 77...

Страница 133: ...e Click on the Start Expert Mode button Fig 4 71 in the LSM 510 switchboard menu The LSM 510 Expert Mode main menu appears on the screen Fig 4 78 Load a Stack or Time Series image which was scanned us...

Страница 134: ...by clicking on File and then on Exit Fig 4 81 The LSM 510 switchboard menu appears on the screen again 4 6 4 Activate User Defined Examination Methods in the Routine Mode Start the Routine Mode Click...

Страница 135: ...appears in the list of User Defined Examination Methods The maximum of 5 methods can be defined as a standard and assigned to a button 4 6 5 Apply User defined Examination Methods in the Routine Mode...

Страница 136: ...imen for examination in the same way as in the Expert Mode Click on the Next button The Routine Mode Image Setup window becomes visible You can optimize the scan parameters in the Routine Mode Image S...

Страница 137: ...Routine Mode Image Acquisition window appears Fig 86 Click on Start to trigger acquisition of the stack If you want to correct the parameters offered click on the Make Z Cut in the Routine Mode Image...

Страница 138: ...Further modifications of the stack parameters can be performed as follows Click on the Use New Parameter button It is possible to select from fixed stack sizes of 10 m 20 m 30 m 50 m and 100 m After...

Страница 139: ...sitioned and focused for scanning The slide rod on the microscope tube has been pulled out as far as it will go Click on the Config button in the Acquire main menu item This opens the Configuration Co...

Страница 140: ...nel select Objective and Frame size for the scan e g X 512 Y 512 scan On the Speed panel enter a scanning speed of e g 7 to start with Start with the following settings on the Pixel Depth Scan Directi...

Страница 141: ...OPERATION LSM 510 Image Optimization B 40 051 e 07 98 4 83 Click on the Channels button This displays the preset parameters of the configuration loaded Fig 4 92...

Страница 142: ...new window Fig 4 93 As a rule the first scanned image Pre Scan is not ideal since the photomultiplier is not matched to the light output More often than not the screen image is dull and needs subseque...

Страница 143: ...1 e 07 98 4 85 4 7 1 Detector Gain Ampl Offset Ampl Gain In the Scan Control window click on the Cont button This starts a continuous scan Click on the Palette button of the image processing toolbar T...

Страница 144: ...ation LSM 510 4 86 B 40 051 e 07 98 In the Color Palette List panel click on the Range Indicator item The scanned image appears in a false color presentation If the image is too bright it appears red...

Страница 145: ...OPERATION LSM 510 Image Optimization B 40 051 e 07 98 4 87 If the image is not bright enough it appears blue on the screen Fig 4 96...

Страница 146: ...thout picture content just show a trace of blue If necessary re amplify brightness with the Ampl Gain slider Do not change the Ampl Gain setting unless the settings made so far are insufficient for op...

Страница 147: ...ative to the detector in terms of X Y Z coordinates contributes substantially to image optimization Requirements to make pinhole position changes visible immediately The image must be scanned by the c...

Страница 148: ...lice display box simultaneously displays the depth resolution corresponding to the pinhole diameter Image optimization can be effected with the Range Indicator or in the Line Scan mode Optimize the pi...

Страница 149: ...en a standard configuration is loaded The automatic adjustment allows the LSM 510 pinholes to be used with any combination of beam splitters Click on the Adjust Automatically button The Requirements f...

Страница 150: ...t back to the factory adjustment Note A change of the pinhole diameter made manually in the Pinhole box will not be activated in the Scan Control box Therefore changes must always be made in the Chann...

Страница 151: ...an Control window Use the Scan Speed slider on the Speed panel to set the slowest acceptable scanning speed The corresponding pixel scanning time Pixel Time and the total scanning time Scan Time are s...

Страница 152: ...menu item click on the Config button This opens the Configuration Control window Select the necessary configuration by clicking the Recording Configurations button selecting a proper configuration and...

Страница 153: ...the scanning process The scanned image appears in a separate window Fig 4 104 As a rule the first scanned image Pre Scan is not ideal since the photomultiplier is not matched to the light output More...

Страница 154: ...f Track panels The channels used are color highlighted The image optimization processes Detector Gain Ampl Offset Ampl Gain Pinhole adjustment Scanning speed and Average must be carried out separately...

Страница 155: ...mposite image Fig 4 105 Now effect image optimization as explained for the single channel mode but separately for each channel Now click on the Display xy button The composite scan image of two channe...

Страница 156: ...nd Shift buttons from the LSM 510 tool bar The Channel Shift menu appears on the screen 2 Select the channels required for processing in the Shift box by clicking on the Ch1 or Ch3 buttons A tick will...

Страница 157: ...mage shows the result of a pixel shift via the Shift function This image change can be stored in the image database via the Save or Save As buttons For applications requiring 3 or 4 channel scanning p...

Страница 158: ...urse all other transmitted light applications like phase contrast differential interference contrast DIC polarization contrast Pol darkfield can also be performed Image optimization must be performed...

Страница 159: ...mages and Stacks Call up an image just scanned or an image from the database Fig 4 109 The image information called up is shown in a window The two columns of buttons headed Select and Display of the...

Страница 160: ...annel so as to highlight or suppress certain image details Fig 4 110 2 Select Zoom The image can be zoomed by various methods Zoom Auto The scanned image is automatically zoomed to the window size Res...

Страница 161: ...om Enlarges the image by factor 2 Zoom Reduces the image by factor 2 Zoom 1 1 Restores an image zoomed in any way to its original size Zoom Mouse Allows you to enlarge reduce an image using the left r...

Страница 162: ...Stacks LSM 510 4 104 B 40 051 e 07 98 3 Select Slice This function allows you to select and view individual slices from a stack Fig 4 112 Select the slices using the slider on the right Example Slice...

Страница 163: ...e area and circumference measurements Furthermore you can enter comments into the image Fig 4 113 Using the paper basket icon you can delete markings and dimensions in the image After an objective cha...

Страница 164: ...for each channel or jointly for all channels Further image processing possibilities can be activated or deactivated alternately using the More and Less buttons Fig 4 114 6 Select Palette This option...

Страница 165: ...ugh the stack manually Using the Speed1 and Speed2 buttons the preadjusted running speed can be interrogated With the Increment function you can define the step width within the stack Only these step...

Страница 166: ...pressed and drag the square to the required position Release the mouse button Zoom Click on a corner of the square keep the left mouse button pressed and set the required size Release the mouse butto...

Страница 167: ...content to the clipboard from where the image can be accessed and copied to other programs applications e g the MS WORD word processor 11 Select Save Allows you to save the scanned image s under the...

Страница 168: ...a different presentation mode such as Split Mode or Gallery 14 Display Split xy In multiple channel scanning the image is seen to be generated in the different channels together with the composite ima...

Страница 169: ...tack Furthermore it is possible to measure spatial distances Clicking on Display Ortho makes section lines and the correlated section projections in the image appear On the right another menu panel en...

Страница 170: ...maximum of n with n standing for the number of slices in the stack produced If you move the cursor into the image window it changes into a crosshairs symbol By dragging this symbol with the mouse you...

Страница 171: ...the image margin no matter which method you used Section of the XZ plane green through the stack above the image Section of the YZ plane red through the stack right of the image Activating the Dist b...

Страница 172: ...ocessing toolbar Fig 4 122 By varying the parameters X Y Z Pitch and Yaw you can position a section plane of any inclination with any point within the stack volume as the datum The resulting position...

Страница 173: ...ous slices of a stack are shown in a tiled presentation of several rows called a gallery in chronological order of their generation A click on the Display Gallery button not only produces the gallery...

Страница 174: ...s you with information on the Z distance of each section slice relative to the first section plane In a time series the temporal interval is shown in seconds Clicking on the Subset button opens anothe...

Страница 175: ...ich you can select various presentations with the Profile Grid and Filled buttons Via the Filled button you can select between monochrome and color presentations In the Channel list box you can select...

Страница 176: ...B 40 051 e 07 98 19 Display Histo The histogram function displays a graph of the intensity distribution of an image as well as information on the frequency of the various intensities separate for eac...

Страница 177: ...ction shows the intensity profile across the image along a freely selectable line In multiple channel mode the intensity profile is shown separately for each channel The intensity curves are shown in...

Страница 178: ...051 e 07 98 You can place markings wherever you like and follow up this line detecting the intensity profile Fig 4 128 Click on the Diagr in Image button to overlay an intensity graph directly on the...

Страница 179: ...ed a pair Of every pair of pixels P1 P2 from the two source images the brightness level of pixel P1 is interpreted as X coordinate and that of pixel P2 as Y coordinate of the scatter diagram The value...

Страница 180: ...98 Fig 4 130 Identical images produce a clean diagonal line running from bottom left to top right because only pixel pairs 0 0 1 1 2 2 etc can occur Differences between the images cause irregular spo...

Страница 181: ...his panel shows the geometric size of the scanned image This function allows you to measure the area of any plane geometric figure within the scan image The function can be activated by clicking on on...

Страница 182: ...and Stacks LSM 510 4 124 B 40 051 e 07 98 Clicking on the Flood fill button paint jar and moving the cursor to the area to be excluded causes the remaining area to be computed and the result indicated...

Страница 183: ...a top and bottom intensity threshold the area lying within this intensity interval can be computed Specify the thresholds either with the Threshold low and Threshold high sliders or with the and butt...

Страница 184: ...the image can be varied using the mouse pointer in the image window Click on the Info button of the image processing toolbar if you want to view print status information and include it in the print pr...

Страница 185: ...OPERATION LSM 510 Analysis of Images and Stacks B 40 051 e 07 98 4 127 Fig 4 134 Assembly of image intensity profile and scan info...

Страница 186: ...s LSM 510 4 128 B 40 051 e 07 98 A layout generated with Prev Preview can be printed by clicking on the Print Print button Clicking on the Print Setup button opens the Print Setup window in which you...

Страница 187: ...ON LSM 510 Analysis of Images and Stacks B 40 051 e 07 98 4 129 24 Display Info Clicking on the Info button all parameters used to generate the image appear at the left image side Fig 4 136 Image para...

Страница 188: ...ith the buttons Depth Cod Projection and Stereo Fig 4 137 a 3D DepthCod Color coded depth map Requirement A stack of images must be available Clicking on the Depth Cod button opens the Depth Coding wi...

Страница 189: ...Preview function permits you to regard the influence of parameter changes in an image window After finding the optimum adjustment using the preview function you have to generate the final version of...

Страница 190: ...f the Projection button opens the Projection window On the Projection panel set the parameters needed for the animation Turning Axis First Angle Number Projection and Difference Angle Fig 4 140 Click...

Страница 191: ...can be animated with the Anim button in the Select bar In addition the Animate dialog box appears in which you can influence the direction and speed of 3D image rotation Fig 4 141 You can browse thro...

Страница 192: ...OPERATION Analysis of Images and Stacks LSM 510 4 134 B 40 051 e 07 98 To view the computed 3D sequence as a gallery on the screen click on the Display Gallery button Fig 4 142...

Страница 193: ...n the Stereo button opens the Stereo Images window The image to be processed appears on the Source panel On the Stereo Images panel set the parameters needed for stereoscopic viewing such as In the Pr...

Страница 194: ...e Apply button The image is built up twice once each for the red and green colors resulting in a stereoscopic image The stereoscopic effect can only be seen with the aid of red green 3D goggles Fig 4...

Страница 195: ...ATION LSM 510 Analysis of Images and Stacks B 40 051 e 07 98 4 137 On clicking on the Apply button the two stereo mates are presented side by side and can be viewed without red green 3D goggles Fig 4...

Страница 196: ...ant database must be opened first Individual images can be loaded using the Import function 4 9 1 Create a new image database Click on the File button of the main menu toolbar This opens another subor...

Страница 197: ...ant to create in the File name text box e g Convallaria If you want to create the image database in a certain folder drive directory click on the arrow button next to the Create in box This opens a dr...

Страница 198: ...lick on the Create button This creates the new image database in the selected drive and directory The Convallaria mdb window appears presenting the opened database with 0 of 0 image entries Fig 4 148...

Страница 199: ...stored If you want to load a database in another folder drive directory click on the arrow button to the right of the Look in box This opens a drop down list box in which you can select from all avail...

Страница 200: ...the image database you can call up images in the following ways to the next image back to the previous image to the last image of the image database to the first image of the image database The image...

Страница 201: ...image database e g Multi Channel 2_0 mdb image series are shown in a tiled arrangement on the screen Fig 4 153 To select one of the images of the series for normal size presentation double click on th...

Страница 202: ...OPERATION Database Loading and Storing of Images LSM 510 4 144 B 40 051 e 07 98 Fig 4 154 Image processing and analysis can be effected via the two column toolbar see section 4 8...

Страница 203: ...images must be given a name and assigned to an existing or new database Save As Stores a previously stored and called up image under a different name Clicking on either of these buttons opens the Sav...

Страница 204: ...image database Enter the name of the database you want to create in the File name text box e g Single Channel 2_0 If you want to create the image database in a certain folder drive directory click on...

Страница 205: ...Recordset box indicates the current number of the image in the image series contained in this database In the Description textbox you can enter for example the configuration of the image In the Notes...

Страница 206: ...in the main menu Fig 4 159 Click on the Import button This opens the Import Image dialog box Select the data medium and the directory where the relevant image is contained Select the image file by cli...

Страница 207: ...ge to be exported Click on the Export button This opens the Export Images and Data dialog box Under Save in select the data medium and the directory to which the image is to be exported Enter a name f...

Страница 208: ...not system_configuration_ mdb LSM Image files lsm Exported images Tiff LSM Tiff BMP Palette files lsm510 Palette lut Filter files lsm510 Filter krn Pinhole setting files lsm510 PH pos Log files lsm51...

Страница 209: ...o language in the Microsoft Word for Windows and Microsoft Excel for Windows products Experience with Microsoft Visual Basic would also be beneficial for macroprogramming of the LSM 510 An Integrated...

Страница 210: ...7 98 Fig 4 164 This window allows you to manage project data and to allocate macros to the buttons in the main window Before you can record or edit a macro you have to create a project as follows Pres...

Страница 211: ...owing buttons are used for recording and running Recording dialog box Start starts recording Stop stops recording Macro dialog box Run runs a macro Recorded macros are stored in main memory first Befo...

Страница 212: ...witch to the Define Buttons dialog box Fig 4 165 Select the button number from the Button box Select the button labelling in the Text editing box Select the name of the project file from the Project b...

Страница 213: ...ssing of the command lines pause Sets a breakpoint in the line with the text cursor Processes a command line and steps into subprocedures Processes a command line and steps over subprocedures Exits th...

Страница 214: ...ser off on Exit in the Shutdown register The lasers will then automatically be switched off when you exit the LSM program 4 11 1 Exiting the LSM program Close all open windows of the LSM program by cl...

Страница 215: ...57 4 11 2 Running down the operating system Move the cursor to the bottom margin of the screen This opens the taskbar containing the Start button Click on the Start button of the taskbar This opens a...

Страница 216: ...e LSM 510 4 158 B 40 051 e 07 98 This opens the Shut Down Windows window in which you can select between Shut down Restart and Login Fig 4 168 Unless already set by default click on Shut down the comp...

Страница 217: ...system writes unsaved data to the disk About 20 seconds after WINDOWS NT has been run down the Shutdown Computer window appears which tells you that you can now turn off your computer Fig 4 169 3 Turn...

Страница 218: ...Lucifer Yellow FITC Cy2 Rhod Cy3 TexRed Laser 458 488 543 HFT 458 488 543 NFT 1 none none none NFT 2 NFT 3 none none none EM 1 LP 475 LP 505 LP 560 EM 2 EM 3 EM 4 FITC narrow band Rhodamine narrow ba...

Страница 219: ...Rhod Cy3 TexRed FITC Rhod Laser 488 568 488 543 HFT 488 568 488 568 NFT 1 none none NT 570 NFT 2 mirror NFT 3 none none plate EM 1 LP 505 LP 585 LP 585 EM 2 BP 505 550 EM 3 EM 4 FITC narrow band Rhod...

Страница 220: ...50 EM 2 EM 3 EM 4 FITC Rhod Rhod Cy5 FITC Cy5 FITC Rhod Cy5 Laser 488 543 543 633 488 633 488 543 633 HFT 488 543 UV 488 543 633 UV 488 568 633 UV 488 543 633 NFT 1 NT 545 NT 635 NT 570 NT 635 NFT 2 m...

Страница 221: ...C Rhod FITC Cy5 Rhod Cy5 FITC Rhod Cy5 Laser 488 568 488 633 568 633 488 568 633 HFT 488 568 UV 488 568 633 UV 488 568 633 UV 488 568 633 NFT 1 NT 570 NT 570 NT 635 NT 635 NFT 2 mirror mirror plate NT...

Страница 222: ...EM 3 EM 4 DAPI FITC DAPI Rhod FITC Rhod DAPI FITC Rhod Laser 364 488 364 543 488 543 364 488 543 HFT UV 488 UV 543 488 543 UV 488 543 633 NFT 1 NT 490 NT 490 NT 545 NT 545 NFT 2 mirror mirror plate N...

Страница 223: ...3 633 NFT 1 mirror NT 545 NT 545 NT 545 NT 570 NFT 2 NT 490 mirror mirror plate plate NFT 3 plate plate plate plate EM 1 LP 560 LP 505 LP 560 LP 650 EM 2 BP 385 470 BP 385 470 BP 385 470 EM 3 LP 505 B...

Страница 224: ...I nb FITC nb Rhodamine nb Laser 364 488 543 633 364 488 543 HFT UV 488 543 633 UV 375 488 543 NFT 1 NT 545 mirror mirror mirror NFT 2 NT 490 mirror plate plate NFT 3 NT 635 Vis EM 1 LP 650 EM 2 BP 385...

Страница 225: ...rror NFT 3 none none none none plate EM 1 LP 385 LP 505 LP 585 LP 650 LP 505 EM 2 BP 385 470 EM 3 EM 4 DAPI Rhod DAPI Cy5 FITC Rhod FITC CY5 Rhod Cy5 Laser 364 568 364 633 488 568 488 633 568 633 HFT...

Страница 226: ...1 NT 570 NT 570 NT 570 NT 570 NT 570 NFT 2 NT 490 NT 490 NT 490 mirror plate NFT 3 plate NT 635 Vis plate NT 635 Vis NT 635 Vis EM 1 LP 585 LP 650 LP 650 LP 650 LP 650 EM 2 BP 385 470 BP 385 470 BP 3...

Страница 227: ...e none none none EM 1 LP 475 LP 505 LP 560 LP 650 EM 2 EM 3 EM 4 FITC Rhod FITC Cy5 Rhod Cy5 Laser 488 543 488 633 543 633 HFT 488 543 UV 488 543 633 UV 488 543 633 NFT 1 NT 545 NT 570 NT 635 Vis NFT...

Страница 228: ...ne none EM 1 LP 385 LP 475 LP 505 LP 560 EM 2 EM 3 EM 4 DAPI FITC DAPI Rhod FITC Rhod Laser 364 488 364 543 488 543 HFT UV 488 UV 543 488 543 NFT 1 NT 490 NT 545 NT 545 NFT 2 mirror mirror mirror NFT...

Страница 229: ...ring readjustment The filters can be rotated in their cells and with the light path being eccentric relative to the filter center the best transmission area of the filter for the respective wavelength...

Страница 230: ...w 4 1 3 with the Allen key Cautiously pull Scanning Module off the Axioplan stand Attach Scanning Module to the baseport of the Axiovert minding the guide pins 4 1 6 and secure it with the M3 hexagon...

Страница 231: ...OPERATION LSM 510 Annex B 40 051 e 07 98 4 173 Fig 4 171 Change over of the Scanning Module...

Страница 232: ...100 g The HRZ 200 is not used if manual coarse focusing is performed To position the objective in relation to the optical z axis the standard xy microscope stage is used The HRZ 200 features a mount f...

Страница 233: ...the step width of the fine focusing stage Use the arrows of the HRZ slider to move the fine focusing stage upwards or downwards in steps Press the O button to reset the display to the value 0 and to m...

Страница 234: ...20 times using the HRZ 200 and that the image information can be obtained up to 100 times faster with the HRZ 200 Select the Z Mode button in the Scan Control dialog The Z Settings window will open S...

Страница 235: ...e The specifications of the stage are obtained only after a heating phase of approx 30 minutes Furthermore the installation conditions for the LSM system must be observed The maximum reproducibility b...

Страница 236: ...rior to switching off the LSM system and the HRZ 200 is then moved to the lowest position to avoid damage of the objective or object by a possible collision The user must refocus after start up of the...

Страница 237: ...hods panel enables you to reach the Routine Mode Microscope Setup window Clicking on the Stage button opens the following window Fig 4 174 This menu enables you to activate both the scanning stage and...

Страница 238: ...y overwriting the displayed value Values between 1 m and 1000 m can be entered for XY Step Size Z Step Size The size of the focus step is set in the same way as the stage steps 0 1 m is the smallest v...

Страница 239: ...between objective and specimen is increased Load Clicking on the Load button lowers the specimen stage nosepiece to make it easier for you to change the specimen or objective Work Pressing the Work b...

Страница 240: ...king on the Stage button in Acquire Fig 4 175 This menu enables control of the scanning stage a session related MARK FIND function is available to you The Stage Position window shows a symbolic specim...

Страница 241: ...are or the joystick If Manual is deactivated the scanning stage can be moved via the software or the joystick All the functions of the Stage Position window are available Move to Clicking on the Move...

Страница 242: ...the table of the session related marked specimen areas The table includes the ordinal number the X position and the Y position Move to Clicking on the Move to button activates the position selected b...

Страница 243: ...d time depending on an action using Trigger Out during time series These actions are Scan Start Stop Bleach Change of Scan Interval end of a countdown or even a mouse click on a button Interface Front...

Страница 244: ...6 ms Speed 1 Trigger Out ca 100 ms Pulse frequency Trigger In 2x pulse width Trigger Out pulse width Valid edge Trigger In Rising edge Trigger Out Falling edge Caution Never apply more than 5 V or neg...

Страница 245: ...er Output 1 2 Trig2O Out Trigger Output 2 3 Trig3O Out Trigger Output 3 4 Trig4O Out Trigger Output 4 5 8 reserved 9 GND Ground 0V 10 Trig1I In Trigger Input 1 11 Trig2I In Trigger Input 2 12 Trig3I I...

Страница 246: ...r checking the laser input in terms of power and noise With the attenuation filter wheel in front of the diode it is possible to attenuate the laser power reaching the diode It is not possible to sele...

Страница 247: ...press Cont the system will scan with the diode as a channel Choose the right amplification of the signal obtained by using the special neutral density filters in front of the diode or and by using the...

Страница 248: ...ondition which has to be fullfilled to use the monitor diode for this purpose is that the dominating source of noise is laser noise The signal of the monitor diode will always be dominated by laser no...

Страница 249: ...r diode channel ChM 1 and one PMT channel Choose the appropriate PMT channel as source 1 in Ratio settings and ChM 1 as source 2 If this numbering is changed inverted the ratio image will show a inver...

Страница 250: ...ation The two images below show the ratio of the PMT and diode signal left and the sum of all signals right The sum image does not contain any information and can therefore be neglected To get a ratio...

Страница 251: ...tuning should be done with the PMT offset if necessary As told before the calculation of the ratio image is very sensitive to different signal offsets in the two channels used As the offset is influe...

Страница 252: ...The use of a lower filter density should be priorized against the use of a high gain value The value of the amplifier gain of both channels PMT and diode should be set to one if possible Because of a...

Страница 253: ...formulas offered when the button R1 1 is pushed The only formula needed for a ratio image with the monitor diode is type 1 S1 n S2 m x y The values for n and m have to be zero as well as the value fo...

Страница 254: ...an is running Any change in the setting parameters of PMT and diode signal will make a new Gain x in the ratio formula necessary If the adjustment of all parameters is finished only the ratio image ca...

Страница 255: ...mage Sequence 5 3 5 1 2 The Image Properties 5 4 5 1 3 Memory Usage 5 4 5 2 User Interface 5 5 5 2 1 Introduction 5 5 5 2 2 Main Window 5 7 5 2 3 Display Window 5 10 5 3 Functions 5 14 5 3 1 Functions...

Страница 256: ...INTRODUCTION TO LASER SCANNING MICROSCOPY Contents LSM 510 5 2 B 40 051 e 07 98...

Страница 257: ...tial images usually called slices in the dialog boxes the spacing between which is equal Image sequences can contain up to 12 bit of image data per channel A sequence slice can consist of up to eight...

Страница 258: ...Properties Every image sequence has its own set of properties They contain the scaling and the scaling units The scaling and its units are required for 3D reconstruction and measurement If a sequence...

Страница 259: ...in window Main window with the Menu the Tool bar and Gallery All general system functions are located here Gallery Normally several images are required in order to accomplish a particular task These i...

Страница 260: ...ndow Dialog boxes All dialog boxes provide three buttons Pressing the OK button executes the function with the defined parameters and closes the dialog window Selecting the Cancel button does not exec...

Страница 261: ...Menu Open Image Opens a file selector dialog to load an image sequence Save Image As Opens a file selector to save an image or image sequence Save Display As Saves the currently shown contents of the...

Страница 262: ...s Adds or subtracts the grey values of two image sequences Add Subtract Contrast Enhances the contrast and brightness of an image sequence Interactive Automatic Linearize Smooth Smoothes an image sequ...

Страница 263: ...Object Features Volume Features Condition Windows Menu Arrange All Arranges the windows automatically Display The current image is displayed in this window Help Menu Content Opens the help for the sof...

Страница 264: ...ltiple channel sequences each channel could have its own base colour The user can set these colours and the weighting for each channel by pressing the corresponding button at the bottom of the window...

Страница 265: ...r functions can be executed while the player is running There are several ways to stop the player by closing the player window by pushing the red Stop button of the player window the window remains op...

Страница 266: ...windows at right Start slice Currently displayed sequential image End slice The buttons in the left group start and stop playback of an image sequence Reverse playback Forward playback Play forward a...

Страница 267: ...efined The weight can be any value between 0 and 200 The colour can be redefined by clicking on the coloured button on the right of the dialog The standard Windows colour selection dialog is opened Th...

Страница 268: ...ed in the Directories list box Use the Drives list box to choose a different drive In case of choosing the TIFF format in the Files of Type box three number characters are always expected before the d...

Страница 269: ...arl Zeiss Vision format the numbers are generated automatically To load such an image sequence KE Images 0 img in the Files of Type box must be selected The window incorporates the usual file selectio...

Страница 270: ...rive Use the list box Files of Type to select the image format Currently the LSM 510 image format lsm and the Carl Zeiss Vision file format KE Images 0 img is supported By choosing the Carl Zeiss Visi...

Страница 271: ...the current Display window contents to a disk or network drive Fig 5 8 Before the execution of this function any image or image sequence can be selected to be displayed From a multichannel sequence a...

Страница 272: ...sequence can be selected to be displayed From a multichannel sequence any channel status on or off combination can be defined The colours of the shown channels can be set with the function Set Channel...

Страница 273: ...sequence can be selected to be displayed From a multichannel sequence any channel status on or off combination can be defined The colours of the shown channels can be set with the function Set Channel...

Страница 274: ...r any other result of a function to the original image sequence The selected channels of Input 1 and Input 2 are copied to Output The maximum number of channels in an image sequence is eight If the im...

Страница 275: ...the selected channels of Input 1 are copied to Output Parameters Input 1 Input image sequence Output Output image sequence Delete All Images This function deletes all images and image sequences from...

Страница 276: ...dd This function adds two image sequences Fig 5 11 The Add tab sheet of the Arithmetics dialog window must be selected If one or both input sequences are multichannel sequence any number or combinatio...

Страница 277: ...determines how a range overflow is handled 1 Wrap No normalization the grey values are displayed modulo 256 4096 If the result is greater than 255 4095 the value 256 4096 is subtracted from it 2 Clip...

Страница 278: ...ut sequences are multichannel sequence any number or combination can be selected The number of selected channels for Input 1 and Input 2 must be the same They will be combined from left to right This...

Страница 279: ...to it 2 Clip Negative values are set to 0 3 Normalize The resulting grey value range is scaled to the range 0 255 0 4095 4 Shift Clip 128 2048 is added to the difference then negative values are set...

Страница 280: ...MICROSCOPY Functions LSM 510 5 26 B 40 051 e 07 98 Contrast Interactive This function allows interactive changes of the contrast of an image sequence Fig 5 13 The Interactive tab sheet of the Contrast...

Страница 281: ...e intensity resolution in bit Normally the result will get the same resolution as the Input sequence A change will be needed if image sequences with different resolutions should be combined Rising the...

Страница 282: ...lly moves the lower boundary the Control key the upper boundary The vertical scale of the histogram is set using the scroll bar The units are percents of the maximum grey value distribution This setti...

Страница 283: ...e The light and dark grey value ranges with a low share of pixels are excluded from the operation by the parameter Threshold The Threshold units are in thousandths of the total number of voxels Using...

Страница 284: ...The Output histogram shows the resulting histogram They are not editable The horizontal axis represents the grey values from 0 to the maximum which is either 255 or 4095 depending whether the input i...

Страница 285: ...e range of all grey values in the Output histogram are the same This function is used to achieve a better view of an image sequence When Skip Black is checked the grey value 0 will not be taken into a...

Страница 286: ...tages of the grey value distribution maximum This setting has no influence to the function Parameters Image Input image sequence Output Output image sequence SkipBlack 0 Grey value black is ignored 1...

Страница 287: ...l sequence any number and combination of channels can be selected Output will only get the selected channels as results The grey value of every pixel is substituted by a weighted average of its surrou...

Страница 288: ...of the morphology of two dimensional images to three dimensions the structural elements are small volumina Literature Bomans M H hne K H Tiede U Riemer M 3D Segmentation of MR Images of the Head for 3...

Страница 289: ...yzed is always the central voxel of the shape The shape type determines which neighboring voxels are used to compute the resulting voxel The following structural elements are available for all morphol...

Страница 290: ...e central voxel to the minimum of all neighboring voxels affected by the structural element dilation sets the grey value of the central voxel to the maximum If the Grey Morphology tickbox is not activ...

Страница 291: ...bulges in the contour of the region Thin connections between regions are eliminated broken borders between regions are connected and small regions disappear The opposite operation first dilation then...

Страница 292: ...If Input is a multichannel sequence any number and combination of channels can be selected Output will only get the selected channels as results The Input image sequence is eroded Count times with th...

Страница 293: ...This function dilates structures in an image sequence Fig 5 21 In the Morphology dialog window the tab sheet Dilate must be selected Dilation makes bright regions larger on a dark background It also...

Страница 294: ...lue shades of the sequence Input If Grey Morphology is not selected the function will distinguish between 0 and non 0 only The result Output will be a binary sequence Parameters Input Input image sequ...

Страница 295: ...urs of the regions are reduced The opening is applied to the grey value image sequence Input Count times with the shape Shape If Input is a multichannel sequence any number and combination of channels...

Страница 296: ...0 051 e 07 98 Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 cross 2 cube 3 cube cross Count Number of recursive operations Grey Morphology 0 Distinguish betw...

Страница 297: ...s of regions are filled in The closing is applied Count times to the grey value image sequence Input with the shape Shape If Input is a multichannel sequence any number and combination of channels can...

Страница 298: ...0 051 e 07 98 Parameters Input Input image sequence Output Resulting image sequence Shape Shape used 1 cross 2 cube 3 cube cross Count Number of recursive operations Grey Morphology 0 Distinguish betw...

Страница 299: ...he Output image sequence Only one channel of a multichannel sequence can be selected as Input Output will always be a single channel sequence The vertical scaling of the histogram can be adjusted with...

Страница 300: ...es outside the defined interval will be segmented If the option Binary is selected then all grey values in the range from Low to High will be set to white grey value 255 in the Output image sequence w...

Страница 301: ...a multichannel sequence can be selected as Input Output will always be a single channel sequence The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the his...

Страница 302: ...e while all others will be set to black grey value 0 If the option is not selected the grey values within the selected interval remain unchanged while those outside the range will be set to black Para...

Страница 303: ...Boolean dialog window must be selected This function is especially well suited for masking images If one or both input sequences are multichannel sequences any number or combination can be selected T...

Страница 304: ...n dialog window must be selected This function can be used to combine binary masks or regions If one or both input sequences are multichannel sequences any number or combination can be selected The nu...

Страница 305: ...roup in the Boolean dialog window must be selected This function can be used to combine binary masks or regions If one or both input sequences are multichannel sequences any number or combination can...

Страница 306: ...an Not This function carries out a bit by bit negation of an image Fig 5 29 The Not tab sheet of the Boolean dialog window must be selected If Input is a multichannel sequence any number or combinatio...

Страница 307: ...ut 2 is higher than 0 then the voxel values are copied from Input 1 to the image sequence Output If the grey value of the voxel is 0 then the voxel value of the Output image sequence is taken over If...

Страница 308: ...jects with a volume within the range MinVolume to MaxVolume are effected To delete objects outside the range the parameter Select must be active If the parameter is not activated objects outside the d...

Страница 309: ...All holes in objects are filled by this operation Holes are structures which have a grey value of 0 and are surrounded completely by voxels with a grey value not equal to 0 It is assumed that regions...

Страница 310: ...nctions LSM 510 5 56 B 40 051 e 07 98 5 3 4 Functions in the View Menu Render Surface This function displays an image sequence according to the gradient shading method Fig 5 33 The Surface tab sheet o...

Страница 311: ...the Start to the End angle definition The other reconstructions are determined through the linearly interpolated intermediate angles The direction of view is determined from the angles as follows The...

Страница 312: ...ine detail Noisy Input sequences must be smoothed function Smooth before rendering otherwise the surface appears rough Parameters Input Input image sequence Output Resulting image sequence Number of V...

Страница 313: ...is the grey value gradient in the Input volume at the surface voxel position It is not the geometric surface normal The grey value gradient is determined from the grey values in a 3x3x3 cube around th...

Страница 314: ...d Fig 5 34 The Alpha tab sheet of the Render dialog window must be selected One or more reconstructions of the input image sequence are computed according to the alpha rendering method This type of re...

Страница 315: ...s determined from the angles as follows The angle Angle Z determines the rotation of the direction of view on the Z axis The angle Angle Y determines the rotation of the direction of view on the Y axi...

Страница 316: ...haded as if illuminated by a light 5 The method can also be applied to visualize pronounced structures within other enclosing structures if the structures have different grey value ranges 6 In this ca...

Страница 317: ...stops accumulation This calculation must be repeated for each pixel of the ray to generate one Output pixel Then for each Output pixel to produce a 2D Output image for the selected view angle Then for...

Страница 318: ...n process produces a mask image that defines the region A region is a group of voxels that touch at the surfaces or at the edges but not at the corners 18 voxel neighborhood This is illustrated by the...

Страница 319: ...ric value distribution of the region Feature parameters Image Region Valid region Data Minimum volume Measurement condition Feature name Feature parameter Measure ment Region filter Image sequence Reg...

Страница 320: ...erized by a number e g volume area or a densitometrical statistic The features can be selected on the Object Features and Volume Features tab sheets Fig 5 37 The scalings and units are taken automatic...

Страница 321: ...feature lists Selecting one of the entries will fill the right list with these features previously selected features will be overwritten The button Select All will copy all features to the list of sel...

Страница 322: ...inor axis of the ellipsoid with the same geometrical moment of inertia as the object Ellipsoid filled EllipseAF Length of the main axis of the ellipse with the same geometric moment of inertia as the...

Страница 323: ...ic Group Name Name Description Mean Densitometric MeanD Densitometric mean value of an object Standard Deviation Densitometric StdD Standard deviation of the densitometric values of an object Minimum...

Страница 324: ...he other one shows the Selected Features Double clicking on items of the left list will add the Selected Features to the right list Double clicking on an item of the right list will remove this item f...

Страница 325: ...051 e 07 98 5 71 Parameters Available Features List of available object features Selected Features List of selected object features Select All Select all available object features for measurement Remo...

Страница 326: ...e Total volume of all regions Volume Percentage VolVolumeP Total volume of all regions in relation to the volume of the image sequence Volume Features densitometric Group Name Name Description Surface...

Страница 327: ...are used to limit the objects to be evaluated e g only objects with defined minimum value All objects are tested against the defined conditions If the conditions are fulfilled the feature values are w...

Страница 328: ...f conditions are combined with the AND expression automatically To remove a condition line double click on it The parameter Minimum Volume defines the minimum voxel volume for the measurement This is...

Страница 329: ...quence if it is multichannel must be selected with the buttons to the right of the parameter The measurement results can be stored to database files These files are tab delimited ASCII files which can...

Страница 330: ...erest is displayed with the Surface Rendering method If Mask is chosen the object is labelled in a pseudo colour in a new image stack Parameters Mask Image Single channel mask image sequence that defi...

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