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- 14 - 

·

 

total number of particles imaged (counted) 

·

 

field of view dimensions (based on a calibration factor for microns/pixel) 

·

 

depth and width of the flow cell 

·

 

total number of frames that were collected 

 

Fluid Volume for AutoImage Mode 

 
The value for Fluid Volume Imaged for AutoImage mode is automatically generated at the end of a run 
using the method shown below under Particle Concentration Calculation.  The value for “Fluid Volume 
Imaged” corresponds to the actual number of milliliters that were imaged for that particular AutoImage 
analysis. 
 

Particle Concentration Calculation: AutoImage Mode 

 

A summary of the following calculation description can be found in Appendix III.  For AutoImage 
Mode analysis, the method to determine particle concentration of an unconstrained camera field is as 
described in the following paragraph. 
 
There are several values that are measured by VisualSpreadsheet that are required for the calculation of 
Particle Concentration (P/mL or PPM): 
 

·

 

Particle Count 

·

 

Area Based Diameter (ABD) of each particle 

·

 

Equivalent Spherical Diameter (ESD) of each particle 

·

 

Total number of camera images taken for the full duration of the analysis 

 
There are also several parameters that are user defined that VisualSpreadsheet uses in the calculation: 
 

·

 

Acceptable range of camera view 

·

 

Flow cell depth (D) in µm 

 
The acceptable range of camera view is dependent on the properties of the digital camera.  Depending 
on the model of the digital camera, the maximum and minimum number of pixels may be different than 
the values used below in the example calculations.  Typical values for camera pixels are 1024 x 768 and 
1280 x 960.  The FlowCAM will arrive with factory default settings that include the correct values for 
the installed digital camera.  VisualSpreadsheet determines the acceptable region within the camera’s 
field of view by the following process.  Note that the acceptable range of the camera can be changed or 
“constrained” by the user: 
 
Left 

 

=  user-specified number (default value =       0 pixels) 

Right   

=  user-specified number (default value = 1279 pixels) 

Top 

 

=  user-specified number (default value =       0 pixels) 

Bottom 

=  user-specified number (default value =   959 pixels) 

 
 
With these numbers specified, VisualSpreadsheet

 

can then determine the height and width of the 

acceptable region with the following calculations: 
 

Содержание FlowCAM

Страница 1: ...FlowCAM Manual Version 3 0 September 2011 Fluid Imaging Technologies Inc 65 Forest Falls Drive Yarmouth Maine 04096 USA Tel 207 846 6100 Fax 207 846 6110 info fluidimaging com www fluidimaging com...

Страница 2: ...2...

Страница 3: ...SPREADSHEET 38 Main Window 48 File Menu 48 Edit Menu 52 Analyze Menu 53 Setup Menu 54 Context 54 Setup Menu Pump 70 Setup Menu Setup and Focus F6 72 AutoImage Mode No Save F7 77 Trigger Mode Setup F8...

Страница 4: ...ification Window Statistics Menu 114 Classification Window Show Menu 114 Right Click Popup Menu 114 Keyboard Commands 115 Appendix I Flow Cells and Tubing Specifications 116 Appendix II Glossary of Pa...

Страница 5: ...n All other trademarks used within this manual are hereby acknowledged Copyright 2011 Fluid Imaging Technologies Inc All Rights Reserved Worldwide Fluid Imaging Technologies customers may reproduce th...

Страница 6: ...g cables or wiring connectors Electrical units should not be operated in hazardous environments Do not touch any damaged non isolated electrical parts This instrument may be harmed by mishandling the...

Страница 7: ...ereto and ii all shipping costs related to the repair or replacement of the Product or any defective Product components for both on site and off site repairs and replacements Any Product or Product co...

Страница 8: ...manual is written to address and describe the configuration of a model VS IV FlowCAM AutoImaging two Fluorescence Trigger channels and one Scatter Trigger channel equipped with a syringe pump It must...

Страница 9: ...ruments Compact and Durable Packaging Ability to Image Particles 2 m to 3 mm in Diameter Fluorescence Detection Providing Additional Selectivity Scatter Detection for Low Particle Concentrations Basic...

Страница 10: ...cube as the flash LED to detect any disturbances in the laser light If either the fluorescence or scatter detector receives a strong enough signal indicating the presence of a particle of interest th...

Страница 11: ...ey provide a critical interface between the software and all other components of the FlowCAM This complex circuit board measures the parameters of the fluorescence and or light scatter signals and pro...

Страница 12: ...e configuration would be an instrument that uses a 488 nm blue laser for excitation a green emission 525 nm Filter set and images with a high resolution black and white camera Additionally most models...

Страница 13: ...lysis mode in which the FlowCAM captures images of the moving fluid at a regular user defined interval Frames Per Second or FPS This mode is used when processing samples with high particle concentrati...

Страница 14: ...meter ESD of each particle Total number of camera images taken for the full duration of the analysis There are also several parameters that are user defined that VisualSpreadsheet uses in the calculat...

Страница 15: ...ion section to view the total number of pixels from left to right and from top to bottom For Depth Setup Context Flow Cell tab Flow cell section for the Flow cell depth value populated from the Flow C...

Страница 16: ...cessing of these previously collected raw images can be useful for testing the efficiency of capturing particles of interest under different context settings Therefore this mode can assist the user in...

Страница 17: ...bove however a camera image of the field of view is captured only when the predetermined fluorescence or light scatter threshold is exceeded by a particle Note that before exiting this mode or while p...

Страница 18: ...one particle per used image To assist the user with determining if the test sample is too concentrated for optimal Trigger Mode analysis VisualSpreadsheet calculates a Particles Per Used Image value...

Страница 19: ...CAM These two cords belong the FlowCAM and FlowCAM LCD Monitor The monitor is usually powered up first If the system is configured with a wireless mouse it should be fully charged and the wireless key...

Страница 20: ...sition Although the laser may now have power it is not activated until the optics door is shut and the user opens VisualSpreadsheet the laser is software activated For other FlowCAM models the power i...

Страница 21: ...all other applications should be shut down during FlowCAM use especially file monitoring applications such as virus scanners which can significantly impact performance If connecting the FlowCAM to an...

Страница 22: ...ls option This is due to the fact that birefringent particles of interest will appear white against a dark background Users can employ the standard tools available in VisualSpreadsheet to remove any c...

Страница 23: ...the pumps are identical Like any other peristaltic pump the C50 uses occlusion tubing to draw the sample through the pump Currently there are three different sized occlusion tubes used to achieve a wi...

Страница 24: ...when 800 and 1000 m flow cells are used This adaptor connects from the input side of the pump to the output side of the flow cell Changing the occlusion tubing is relatively simple The colored blue c...

Страница 25: ...s the pump in prime mode until the button is pushed a second time Note that the direction of flow in prime mode is the same direction that has been set with the toggle switch The speed control is the...

Страница 26: ...7 FC600 or Greater 9 7 6 C57 FC600 or Greater 10 8 7 C57 FC600 or Greater 11 8 8 C57 FC600 or Greater 12 9 9 C57 FC600 or Greater 13 9 10 C57 FC600 or Greater 15 0 Dial Setting Pump Tube Flow Cell Flo...

Страница 27: ...le See Section Four for more information regarding software control of the syringe pump During the set up of the syringe pump tubing lengths must be considered Ensure the tube lengths above and below...

Страница 28: ...e shorter and straighter the tubing the better Sample Funnel Using a six inch ring stand measure and cut the tubing upstream of the flow cell Lower the funnel holder ring all the way down around the t...

Страница 29: ...29 Figure 13 An example of the pipette tip holder installed on the FlowCAM Figure 14 Tubing to Funnel Connection and connection to Pump for a Benchtop FlowCAM also see Figure 15 below...

Страница 30: ...rate results especially when utilizing the FlowCAM for counting concentration determination the flow cell needs to be free of bubbles Bubbles can be removed from smaller flow cells 50 100 300 m by pin...

Страница 31: ...ttings Layout Status Bar Show Status Bar The Status Bar is color coded to match the color line on the objective The colors are black 1 2X Blue 1X grey 2X red 4X yellow 10X and green 20X Figure 17 Exam...

Страница 32: ...on adjustment of the flow cell Fine adjustments and positioning of the flow cell is possible by use of the X and Y axis positioning screws Figures 18 19 This adjustment should be done for example to m...

Страница 33: ...l the light intensity can be accessed from two different menus in VisualSpreadsheet The first menu is located via Setup Context Camera tab and Timing tab The second menu is accessed by selecting Camer...

Страница 34: ...st the intensity of the light passing through the flow cell is a manual method To center the column of light within the field of view one can manually re position the LED flash by adjusting the screws...

Страница 35: ...Spreadsheet and collect data VisualSpreadsheet is described in detail in Section Four When finished with data collection and image analysis follow the Shut Down procedure described in the next section...

Страница 36: ...51 When the pump is not in use unclip the occlusion tubing This will extend the life of the tubing and provide the most consistent results o At the minimum change the occlusion tubing every month o I...

Страница 37: ...in observed particles even when liquid is still moving through the flow cell Field of View FOV Flow Cells It is recommended to replace a field of view flow cell if o the seal between the stainless st...

Страница 38: ...JJJ hhmmss xxx where JJJ is the Julian day 1 365 and hhmmss represent the hours minutes and seconds for the start of the experiment Finally xxx is the filename extension for the stored file The automa...

Страница 39: ...is generated with each list file It records the context information that was in effect when the list file was generated or when the experiment was run The name of the context file uses the same commo...

Страница 40: ...he FlowCAM typically utilizes a Core 2 Duo 2GB RAM and 500GB HD larger hard drives are preferred for image storage The Operating System presently used is Windows 7 but older systems employ Windows XP...

Страница 41: ...41 Main Window and View Window Examples Figure 22 The Main Window for VisualSpreadsheet left and an example of the separate View Window right including the Particle Properties Display...

Страница 42: ...e main sections highlighted and described for reference Summary Statistics Filters Cumulative Stats Context Summary tabs Analysis Start End Times Particle Counts Main Window Menu Toggle Button ESD ABD...

Страница 43: ...A check mark indicates the type of graph currently being displayed See Figure 24 for an example of selecting a Histogram or Scatter Plot Figure 24 Right clicking an interactive graph to display the ty...

Страница 44: ...e particles graphed in the selected area Alternatively you can open the View Window by Edit Open View Window F2 Using this alternative method the View Window will only display the particles currently...

Страница 45: ...the length of the horizontal line or rectangle selection area and the particles in the selection area are shown in the View window Scattergram Plots Left click and drag the mouse over the region of i...

Страница 46: ...ry Statistics grid is configured via Preferences Main Window Settings Summary Statistics tab The user can select which calculated values columns and which Summary Statistics rows are displayed in this...

Страница 47: ...47 Figure 30 An example of the Cumulative Stats tab from the Main Window Figure 31 An example of the Context Summary tab from the Main Window...

Страница 48: ...opened in the Main Window can be seen in Figure 23 If VisualSpreadsheet is directed to open a List file that was collected with a different magnification than the currently selected magnification an...

Страница 49: ...ginal data file is not overwritten To protect the original List file the file name entered in the Save List As dialog box must be different then the name of the List file currently open Otherwise the...

Страница 50: ...Open button If the Cancel button is selected then the file open dialog box closes and the Library Window remains open without any library files open More than one library file can be open at the same...

Страница 51: ...ction also creates a file named summary_export csv in the working directory for the List file that is currently open The particle or summary data export functions work on the data that is currently in...

Страница 52: ...graph Note that the View Window must be closed to enable this menu option Particles deleted using this menu option are not permanently deleted from the original List file But once the displayed List...

Страница 53: ...ects images for a pre set time period for a number of particles or until the user terminates the analysis File Processing Mode The File Processing Mode is used to process raw images full field of view...

Страница 54: ...ludes settings for the camera fluorescence gains and thresholds and other various parameters The settings are stored in a file with ctx file extension within the folder containing all the files for a...

Страница 55: ...a file open dialog box The file open dialog box is displayed with the ctx file extension Select an existing Context file and click the Open button Using an existing Context file can save time over man...

Страница 56: ...age using a 3x3 pixel neighborhood For each neighborhood the process is as follows Dilation if any of the center pixel s neighbors are white considered part of a particle in the thresholded image then...

Страница 57: ...Save Raw Camera Images is enabled Due to the large size of each raw image caution should be used when selecting a value for this parameter to avoid reduced frame rates and excessive disk space usage P...

Страница 58: ...hart of the flow cell types which are also the part numbers for the flow cell and specifications Tubing The Tube inner diameter value will automatically populate with the diameter once a flow cell typ...

Страница 59: ...o be used to perform the camera background calibration an error message will appear If the volume needed to calculate the background calibration image exceeds the entered sample volume then the analys...

Страница 60: ...se lengths When adding the sample into the introduction port pipette tip or funnel ensure that the bottom or leading meniscus of the sample is located between the top of the flow cell tube and the bot...

Страница 61: ...te or neutral grey colored background The best way to accomplish this goal is to open and view the Setup and Focus window while adjusting the parameters Refer to the following illustration for a guide...

Страница 62: ...e frames per second to use while running in AutoImage Mode The AutoImage Rate is typically used in conjunction with the pump flow rate to minimize multi imaging of particles Flash Camera Delay This pa...

Страница 63: ...s images of particles that fall within this filter s size range For example a raw camera image may contain hundreds of particles with ESD values that range from 1 90 m but the user is only interested...

Страница 64: ...s applied after the Basic Size Acquisition Filter Any particles meeting the Basic Size Acquisition Filter requirements are then subjected to the Advanced filter When enabling this feature the user wil...

Страница 65: ...a classification or export a classification summary at the end of a run The user can also Find and select a Classification Template previously constructed see the section describing Classifications f...

Страница 66: ...nformation is contained Run Mode Priming Method Flow Rate Recalibrations Stop Reason Sample Volume Aspirated Sample Volume Processed Fluid Volume Imaged Efficiency Particle Count Images Total Used Per...

Страница 67: ...he analysis ends when the specified time limit is reached after user set value collage files The analysis ends when the specified of collage files have been collected after user set value camera image...

Страница 68: ...rigger Mode Channel 1 and Channel 2 These are the two channels that utilize PMTs to detect fluorescence signals from particles in the sample that fluoresce see Figure 2 letter B The standard configura...

Страница 69: ...fety section at the beginning of this manual for a listing of precautions regarding laser safety Fluorescence Scale Linear or Log The user can choose to report the peak and area values of the fluoresc...

Страница 70: ...e PMTs and scatter detector are recorded for each required channel using the same sampling cycle as previously described After the measurements are complete the flash LED is illuminated and the camera...

Страница 71: ...e Pump option Syringe Pump Controls These functions allow the user to change the size of syringe manually prime the system and use the automated flush function to clean out the system If a syringe pum...

Страница 72: ...d cycles The Current Cycle is displayed and the process can be stopped at any time by clicking the Cancel button The checkbox labeled Automatically send sample to waste bottle is an optional setting t...

Страница 73: ...eters that are accessed via Setup Context Camera tab The benefit of accessing these camera settings from the Setup and Focus Mode window is that any adjustments and changes are displayed in real time...

Страница 74: ...ler can be adjusted via Preferences Display Settings Image Windows tab Ruler section Note that the color option in the Preferences menu will only change the color of the Ruler in the Collage Window To...

Страница 75: ...o 255 Show fluorescence scatter Displays the fluorescence and or scatter value for FlowCAM models configured for Trigger Mode When enabled this parameter will not be displayed on the Setup and Focus w...

Страница 76: ...d run time if displayed are also reset Raw Pixel Intensity This advanced tool can be used as a guide to help set the illumination uniformity of the image when adjusting either the LED adjustment knob...

Страница 77: ...se and function of this tool The Masked Image Pixel Intensity is the same as Raw Image Pixel Intensity except that the intensity values have the intensity of the background calibration image subtracte...

Страница 78: ...tration and trigger thresholds File Processing Mode No Save The software processes previously collected and saved raw images to generate a new data set using the current context settings Similar to Fi...

Страница 79: ...on which is Close which closes the Time Series Graph The Settings Menu contains two functions The first one is Configure which allows the user to set up how the data will be displayed on the graph On...

Страница 80: ...ibration Factor Typically more accurate results are obtained using the calculations and methods of the current version 3 0 of VisualSpreadsheet Selecting No will result in the particle properties and...

Страница 81: ...w as determined by the above selections can be saved as a template denoted by a page layout file type of plf by using the Save Page Layout button During future use the Load Page Layout button can be u...

Страница 82: ...han D10 Similarly the D10 value for volume is the value such that 10 of the sample particles have a smaller volume than D10 and 90 have a larger volume than D10 D 4 3 ABD and D 4 3 ESD Values These va...

Страница 83: ...Count to be used to determine the cumulative distribution from which percentage points are displayed in the Cumulative Stats grid on the Main Window Fields The user can select Diameter Aspect Ratio or...

Страница 84: ...when data are exported in spreadsheet format Images If selected when image files are saved the particle ID numbers are shown with each particle image in the saved file Export Settings Data Fields Fie...

Страница 85: ...user The other section Image Acquisition Delay contains parameters that typically should not be adjusted by the user because they are factory set to default values Logging When this function is enabl...

Страница 86: ...ered and the Apply button is selected a warning will be displayed to indicate that a critical value is about to be modified Select Yes to continue or No to cancel Help Advanced Tools Menu Advanced Tim...

Страница 87: ...Firmware function is an advanced tool that is for use by Fluid Imaging Technologies trained personnel It is not recommended that the FlowCAM user utilize this function unless directly instructed to do...

Страница 88: ...with the symbol displayed Deleting or filtering particle images using the tools and functions available in the View Window will not update or change the statistics in the Main Window The Main Window w...

Страница 89: ...atus Bar at the bottom of the View window provides information about the following statistics of the images Saved Modified The word Saved in the first section of the Status Bar indicates that the curr...

Страница 90: ...Interactive Graphs in the Main window For example if a filter is currently applied only those particles that meet the filter conditions will be saved Save Image The Save Image command saves the image...

Страница 91: ...taken to save the results of filtering sorting and deleting if desired before closing the View window View Window Edit Menu The edit menu is used to select and deselect particle images and for deletin...

Страница 92: ...lear Filter Scores If images have been sorted or filtered in any way they receive a filter score Selecting this option removes these score s so that a new sort filter can be applied View Window Sort M...

Страница 93: ...osition this will have the opposite effect the largest particles are displayed in the first collage and the smallest are displayed in the last collage Filter Score The Filter Score is a dynamic derive...

Страница 94: ...in the training set A natural choice for the cutoff value is the Filter Score of the highest scoring particle in the user provided training set This is the default value that VisualSpreadsheet uses fo...

Страница 95: ...the Filter Min and Filter Max are entered by the user Selecting the More Options button will reveal the additional advanced options Filter Persistence Once constructed filters can be saved and then r...

Страница 96: ...as also defined Auto Select Particles Normally a Statistical filter will use the highest scoring particle part of the training set as the cutoff score for selecting particles that match Unchecking thi...

Страница 97: ...elected by the previous analysis as additional input to the next analysis or by manually adding or removing input particles between iterations by selecting deselecting particles with the mouse The Sta...

Страница 98: ...eadsheet seeks out the collage files on disk for updating the display To speed operations where viewing images is not always necessary the user can disable image display and easily re enable it again...

Страница 99: ...empting to improve the overall analysis data and statistics Classified Images Normally when the Classification window is displayed any particles that are already part of a Class are not shown in the V...

Страница 100: ...to any Library currently open Note that the Library Window must be open with at least one Library open in it The particles remain selected and if this function is used again without deselecting the p...

Страница 101: ...ary Window Tool bar is just above the image collage area It functions in the same manner as the View Window Tool bar The green arrow buttons are used to navigate through the various pages of collage i...

Страница 102: ...rary Selected Indicates the number of particle images currently selected The particles selected can be from multiple image collage pages The Selected number is the total for all image collage pages So...

Страница 103: ...tly selected Library Tab in the Library Window is saved to disk using a different Library file name The name of the selected Library appended with _copy is automatically provided as the File name in t...

Страница 104: ...layed page The red border is removed from any particle images currently selected Delete Images Selected Deletes any particle image that is currently selected images with a red border Note that with th...

Страница 105: ...s is the order of acquisition and corresponds to the equivalent numbered row in an exported data spreadsheet The Sort Menu allows you to change the order in which the particle images are displayed in...

Страница 106: ...re not sorted together by the use of various shades of colors If parameters as seen in the Particle Properties window are lightly colored then their differences from filtering values were minimal if t...

Страница 107: ...bar The green arrow buttons are used to navigate through the various pages of collage images and the blue arrow is for Ascending Descending order To navigate to specific collage page click in the text...

Страница 108: ...eing shown in image collages for the currently selected Class tab If the Delete Images Selected or Delete Images NOT Selected menu commands are used then the Total will be decreased by the number of p...

Страница 109: ...s any changes for the currently open Classification to the list file that is currently open Save Classification As The currently open Classification is saved to disk using a different Classification n...

Страница 110: ...any particle images currently selected Select All This Page Ctrl F3 Selects all particle images on the currently displayed page This will place a red border around each particle image Deselect All Thi...

Страница 111: ...yed in one row of tabs blue scroll arrows appear after the last tab click on the previous or next arrow to scroll through the tabs Tabs cannot be dragged to rearrange their order they must be opened i...

Страница 112: ...d to be deleted Export Class Data This menu command exports the particle property values for each particle image in the currently selected Class tab in the Classification Window Export Class Summary T...

Страница 113: ...that classes are opened Clear All Classified Particles This function will remove the particles selected by the Auto Classification function and return them to the View Window Classification Window So...

Страница 114: ...fication Window Statistics Menu Summary Statistics This window provides statistics on the particle properties for particle images being viewed in the currently selected Class tab Classification Window...

Страница 115: ...e open in the Classify window Keyboard Commands Selecting Multiple Particle Images To select a group of particle images within the Classification Window press and hold the Ctrl key click to the left o...

Страница 116: ...FC100 100 2000 0 16 10X or 20X FC200 200 2000 0 16 10X or 4X FC300 300 3000 0 16 4X FC300FV 300 1500 YES 0 16 4X FC600 600 6000 0 40 4X or 2X FC700 700 7000 0 40 4X or 2X FC800 800 8000 0 50 4X or 2X...

Страница 117: ...Average Red Average pixel value of the red color plane real 0 255 255 is intense red Calibration Factor The conversion factor from pixels to microns units are microns per pixel real fixed for each ins...

Страница 118: ...th and Perimeter 2 length breadth real 1 1 is the value for a filled circle or square larger values are for elongated particles Feret Measurement The perpendicular distance between parallel tangents t...

Страница 119: ...Area Area of the peak of the scatter detector measured over the width real 0 Scatter Peak Peak value read from the scatter detector real 0 Scatter Width Sample width of scatter detector values above...

Страница 120: ...d 1280 x 960 The FlowCAM has factory default settings that include the correct values for the installed digital camera VisualSpreadsheet determines the acceptable region within the camera s field of v...

Страница 121: ...6 Total Volume Imaged m3 Particle Count 1 x 1012 m3 P mL X Total Volume Imaged m3 1 mL To locate the values used in the above calculations from the Main window For Height and Width access Setup Contex...

Страница 122: ...les or organisms Additionally you would use Trigger Mode if you have a very dilute sample and you wish to ensure that you take a picture of each particle rather than risk missing particles or capturin...

Страница 123: ...elect Trigger Mode 15 Input your desired stop criteria and click OK 16 Save the run in an appropriate location with any file name you wish and click Save 17 Allow run to finish Repeat above steps for...

Страница 124: ...refore the same particle properties related to the trigger event such as CH1 Peak are assigned to all particles within that raw image This is the main reason that your sample should be as close to One...

Страница 125: ...es below to determine the appropriate bead size mix and the corresponding Basic Size Acquisition Filter minimum and maximum diameters Objective Bead Sizes m 20X 5 10 20 10X 10 20 50 4X 20 50 100 2X 50...

Страница 126: ...cell Stop the run before the meniscus enters into the camera s field of view to prevent the erroneous imaging of bubbles Interpretation of Results Each of the six filters when selected will display t...

Страница 127: ...e flow cell tubing as indicated in the graphic below The length of tube from the top of the flow cell to the bottom of the pipette tip is known as the dead volume This volume of liquid dilutes the con...

Страница 128: ...and via the Statistics menu item in the View Window Interpretation of Results The Particles mL value will be displayed on the Main Window and via the Statistics menu item in the View Window Use the T...

Страница 129: ...Procedure Objective Bead Size m Listed Count P mL FlowCAM Count P mL Pass Fail 2X 4X 10X 20X If a particular objective was not required available for testing enter N A Notes reagent lot numbers etc Te...

Страница 130: ...he FlowCAM not applicable to all FlowCAM models 2 Turn on the FlowCAM using the power switches located on the rear and front of the instrument 3 Wait for the operating system to fully load 4 Set up th...

Страница 131: ...ribution and sample dilution to ensure that the best possible data are obtained Use the fine focus adjustment knob to adjust the focus until the sample appears to look the best possible Setup and Focu...

Страница 132: ...Save mode Determine if the FlowCAM is identifying particles of interest to you and segmenting them correctly Close the No Save run and make changes in the Context Settings and repeat as needed Insure...

Страница 133: ...4X N A 10X Collimator 20X Collimator Flow Cell Objective Recommended Flow Cells 1 2X Custom FC4000 FC3000 FC2000x6 FC2000x4 FC1000FV FC1000 1X Custom FC4000 FC3000 FC2000x6 FC2000x4 FC1000FV FC1000 F...

Страница 134: ...C FOV10 4X QCFC600 QCFC300 QCFC FOV10 QCFC FOV5 10X QCFC300 QCFC FOV10 QCFC FOV5 20X QCFC300 Pump Tubing C50 51 Objective Recommended Pump Tubing for C50 51 1 2X C57 Clear 1X C57 Clear 2X C 57 Clear C...

Страница 135: ...ml 4X NF505 5ml NF502 2 5ml NF501 1ml 10X NF502 2 5ml NF501 1ml 20X NF502 2 5ml NF501 1ml Nitex Filter Size Objective Recommended Filter Media Pore Size 1 2X NM 1000 1000 m 1X NM 1000 1000 m 2X NM 100...

Страница 136: ...inary collage images and raw camera images for this run Use the binarization overlay to demonstrate that the software did not use the pixels at the center of the image of a bead because those pixels a...

Страница 137: ...xels that comprise the thresholded particle in a solid circle ESD Equivalent Spherical Diameter is the mean feret measurement of the particle based on 36 sample measurements conducted every 5 Q Should...

Страница 138: ...ing them as filters Note that when using a library for filtering it is recommended that you only have 10 15 organisms in it to avoid introduction of error Then after refining these library selected cl...

Страница 139: ...ps 10 17 Q How do I sort a List File using the Classification function A Follow the procedure below to sort a List File using the Classification function Notes on Classifications Auto Classification f...

Страница 140: ...teps in section III below 4 Main Window Edit Show All Data 5 Main Window Edit Open View Window 6 View Window Show Classified Images should be Unchecked 7 View Window Manually classify any particles th...

Страница 141: ...73 Camera Flash Duration 34 Camera Gain 34 Camera Setup 33 Camera Type 86 Capture Context Setting 56 Setup and Focus Mode 73 Change Syringe 71 Changing Graph Type 43 Channel 1 68 Channel 2 68 Classes...

Страница 142: ...rate 59 FlowCAM Summary 9 FlowCAM XPL 22 Fluid Volume 58 59 Fluidics 10 Fluorescence Scale 69 Fluorescence Scatter Setup and Focus Mode 73 Flush System 72 Gain Context Setting 61 Gamma Context Settin...

Страница 143: ...9 12 36 Pump Controller 71 pump speed 18 Raw Image Pixel Intensity tool 77 Raw Pixel Intensity Setup and Focus Mode 76 Recalculate 80 Recalibrate Setup and Focus Mode 76 Recalibration 68 Recalibratio...

Страница 144: ...Filter 96 114 Statistical Filter Behavior 96 Statistics Menu 97 105 114 Status Bar 31 81 89 102 107 Stop Context Setting 67 Stop Conditions 67 Summary Statistics 46 97 114 Summary Stats tab 46 Syring...

Страница 145: ...145...

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