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ImageStream System Software User`s Manual

1. Classification parameters limit the number of viewable objects In the cell detection window either click Ignore all enabled or turn all classification parameters off and toggle image view mode to All Set parameters to include desired cell images Insufficient illumination Make sure the excitation and or scatter laser is turned on and set to the proper intensity setting Make sure the brightfield lamp is turned on and set to the proper intensity setting TROUBLESHOOTING 105 SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Core stream is outside the Manually find the core stream Turn the laser to objective s field of view 100 mW core track left or right in 10 m incre ments to find the core and then manually adjust focus Squelch setting is too Set squelch to 0 Gradually increase the squelch high value to retain desired images and eliminate debris No cell images Imaging is paused Click Resume Image view mode not set Toggle image view mode to Cells or All to cells Cells are classified as In the cell detection window either click Ignore debris all enabled or turn all classification parameters off and toggle image view mode to Cells Set parame ters to include desired cell images Cell Classifiers Ignore all enabled box Right click in the cell detection window and are not working is checked uncheck the Ignore all enabled item Cells are not masked In the Cells view
2. vis des microbes Le Niveau de s curit biologique pour l instrument est de niveau L1 SPARE PARTS LIST 8 The instrument contains no serviceable parts Only Amnis trained technicians are allowed to repair maintain and set up the alignment of the laser beams INFORMATION AND SAFETY CHAPTER 2 Introduction This section describes the technology used in the ImageStream system the requirements for cell samples and the fluidics system It includes the following subsections e Introduction to the ImageStream e Technology Overview e Basic Illumination Optics and Image Collection e Hydrodynamic Focusing SpeedBeads and Cell Samples INTRODUCTION TO THE IMAG ESTREAM The Amnis ImageStream is a bench top multispectral imaging flow cytometer designed for the acquisition of 12 channels of cellular imagery By collecting large numbers of digital images per sample and providing numerical representation of image based features the ImageStream combines the per cell information content provided by standard microscopy with the statistical significance afforded by large sample sizes common to standard flow cytometry With the ImageStream system fluorescence intensity measurements are acquired as with a conventional flow cytometer however the best applications for the ImageStream take advantage of the system s imaging abilities to locate and quantitate the distribution of signals on
3. Exclusion of fluorescence images with saturated pixels by setting a Raw Max Pixel upper limit of 4095 counts in the appropriate channel e Exclusion of small debris by setting an Area lower limit in the brightfield or scatter channel OPERATING THE IMAGESTREAM USING INSPIRE 25 e Inclusion only of cells with positive fluorescence by setting a Raw Max Pixel lower limit in the appropriate channel Use the following procedure to set cell classifiers 8 Click Cell Classifier icon to launch the Cell Classifier window Cell Classifier Parameters Channels to Collect Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 M Collected M Collected M Collected Collected M Collected Collected Area Lower Limit Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 ar Ar Ar 4r 4 ims5 Area Upper Limit Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 r arj A FF o4 r Sar Sar e Right click to select the classification feature s Selected feature s appear as a new row in the popup window e Enable the feature classifier s by checking the box es in the desired channel s and type in the desired threshold value s e An overlay mask can be shown on the object images Yellow for debris green for cells and purple for SpeedBeads e Note Select Ignore All Enabled on the Context menu to stop cell classifi cation without losing the classifier values To verify that classifier has wo
4. POSSIBLE CAUSES RECOMMENDED SOLUTIONS Flow speed sig nal disappears No Images Clogged flow cell Clogs may occur at the taper between the flow cell and the cuvette Verify that the flow cell taper is actually clogged by doing the following Remove the waste tank and verify that sheath is dripping from the waste line while running If no drops appear return the sample and run the Sterilize script If this does not remove the clog call Amnis service Sheath incompatibility with sample The Autofocus Flow Speed system has stopped operating Verify you are using the appropriate sheath solution refer to the Preparing the ImageStream for oper ation chart For experiments using beads or con taining surfactants dH2O sheath is optimal For running cells Ca Mg free PBS is optimal Toggle the IR laser off and then on by clearing checking the FSP Enabled box Pumps are empty If sheath syringe is empty load sheath then dual prime If the bead pump is empty choose Flush and Load from the Beads menu Air or clog in the sample valve See solution for Air or clog Clogged flow cell Camera is not running See solution for Air or clog Click Run Setup If the camera is already running click Stop to stop the camera and then click Run Setup Imaging is paused Image view mode is set to debris Click Resume Toggle image view mode to All Cells or Beads
5. EDF is a novel technique used in a variety of applications including FISH spot counting where having the entire cell in optimal focus is critical to obtaining accurate results There are two steps to utilizing the 16um EDF first images must be acquired with the EDF element in place and second the data must be deconvolved using the EDF kernel prior to analysis This calibration of the EDF element generates and saves the kernels used by IDEAS to deconvolve the imagery Calibration of the element is done on installation and should be repeated by Amnis service when any optical changes are made ES EDF Excitation Utility Parameters Num Settings Num samples Total Range o a oe 3 2 a A 5 n Database Setting ASSIST EDFCalib_Value ASSIST EDFCalb_Kemelwidth ASSIST EDFCalib_KemelHeight ASSIST EDFCalib_ExKermelKOffset ASSIST EDFCalib_ExKemelY Offset ASSIST EDFCalib_ExCh1Kemel_Default ASSIST EDFCalib_ExCh2Kemel_Default Channel Of Interest Focus Position um Saved Value Result Value 0 00 32 32 10 22 000000 000000 TO PERFORM THIS CALIBRATION 1 Verify that the instrument has been initialized ASSIST has been run and the instrument has passed the set of calibrations and tests run with Start All 2 Stop the fluidics by clicking stop on the Setup page 3 Deselect the Ignore All Enabled option in the cell classifier if necessary 4 In the file menu choose Open template and select the template named EDF
6. It is critical that you run this sample first to establish the instrument settings DO NOT change laser settings for the experi ment once established on this sample if you are using dyes that are excited by more than one laser Vie Iretumert Sample Lesds Hep run setup Run segure Return Sample CuslPrme Prime Beads CR cht ae oe Cha as c When prompted place sample vial into the sample loader 50 ul of sample is loaded ensure that the vial contains a minimum of 50ul 2 Inthe file menu choose Open Template if an experimental template exists or manually set up the instrument to create one Note Application specific instrument settings can be saved in a template and used to facilitate instrument setup but it is recommended that you verify the appropri ateness of the settings for the specific experimental run 3 Choose the objective under Magnification 24 OPERATING THE IMAGESTREAM USING INSPIRE 4 Turn on each laser used in the experiment by clicking on the radio button for the laser Set the laser powers so each fluorochrome has peak intensities between 100 and 900 counts as measured in the dot plots and there is no saturation Excitation Lasers OrV OMt Irtarsity rn nm Cp foo em amp no 3 sam B foo 75m GP hw 5 Select Ch1 for Brightfield for a one camera system Ch1 9 for a two camera system and click Set Intensity 6 Select EDF collection if desired See Usi
7. Make sure the desired velocity setting is set to 60 mm sec or lower Click the Lock on button to acquire the desired core stream velocity Autofocus is not tracking Reset the focus position If the cells come back into properly focus turn Autofocus tracking on If the cells grad ually become defocused as the focus position moves then Autofocus tracking is not working Run with Autofocus tracking off and call Amnis service Flow speed is The sample is clumpy or Filter and or concentrate the sample not stable is not at a high enough concentration There is a clog or air bub See solution for Air or clog in the sample syringes ble in the system or flow cell Insufficient fluid volume Fill the waste tank with enough fluid to immerse in the waste tank the waste line outlet Flow speed Insufficient fluid volume Fill the waste tank with enough fluid to immerse changes rhyth in the waste tank the waste line outlet mically Fluidics Leak in the fluidic system Call Amnis service respond slug gishly Actual velocity Fluidic script is running Wait for core stream to form reading appears and or core stream has frozen not yet formed Autofocus Flow speed Turn the IR laser on by checking the FSP Enabled system is not operating box Advanced Flow Speed tab Toggle the IR laser off and then on by clearing then checking the FSP Enabled box Event rate slows Cells have settled in the Cells settle in the line
8. Select Defined button or e All In this example column 1 is selected Well Plate Definition D Start Plate Definition Name EE Customize Well Plate Select by Color E E mE Select Defined Delete Selected Wells gt lz o mlolole gt KEEKEEKE eeeeeeoe er eeeeeeo eeeeeee o eeeeeee Os eeeeee gt eeeeeee ol eeeeeee eeeeeee Oe eeeeeee Oe eeeeeee ol 6000606606 7 You can edit values for some of the Custom and many of the Standard parameters You can do this for all selected wells or for individual wells For example if you want to collect 1000 events for the selected wells type 1000 in the Apply to selected box below the Cell Count heading If you want to only apply this to a single well type this value in the Cell Count box corresponding to that well Below is an example of a Well Plate Definition using several parameters and show ing only defined wells 35 OPERATING THE IMAGESTREAM USING INSPIRE ee Flename Wel Date Cor Output File Path le Template Fie ro Stan Treatment Comments Patierttt Dose Time Include in filename ro ro ro no ver yee m ves yes yer Apply to selected T C Documents and Settings amnis Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 043010 WB CDASFITC it Goris com CD4SF TNF Al worn TE Blue C Docurents and Setting amrie Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 04
9. Setup tab and establishes a core e Load Loads sample into tubing Note used for service do not use for run ning samples Prime Sample Rapidly delivers the sample to the flow cell Return Sample Empties the syringe returning the loaded sample to the sample vial This results in some loss and dilution of sample e Beads menu Run fluidic scripts specific to the bead pump Beads Flush and Load Prime Beads Flush and Load Backflushes the sample and bead lines and rinses the bead syringe with sheath fluid three times and primes the beads at the end of the process to refill the flushed line to the flow cell Use this option to reload the SpeedBeads from the 15 ml conical tube Prime Beads Rapidly delivers beads to the flow cell e Help menu Help Help Topics About ImageStream About ImageStream Access the current INSPIRE version number with the About ImageStream option 42 INSPIRE M SOFTWARE OVERVIEW HE AboutBox amnis INSPIRE for the ImageStream ACTION BUTTONS The text buttons above the imaging area in the INSPIRE screen are used to run the camera image acquisition and to run fluidic scripts which are automated fluidic routines The action buttons include the following Run Acquire Stop Flush Lock Load Return Sample Dual Prime Prime Beads Initialize Fluidics Sterilize e Run Setup Runs the camera allowing you to view imagery and feature data without ac
10. verify that cells of interest are being masked appropriately Imaging is Sample concentration is Make sure the sample concentration is 1 x 107 cells intermittent or low per ml As a guide a sample concentrated to 107 appears frozen cells ml runs at 100 cells per second when the core and object stream is 10 microns in diameter traveling at 60 acquisition rates mm sec are erratic Classification parameters In the cell detection window either click Ignore limit the number of all enabled or turn all classification parameters off viewable objects and toggle image view mode to Cells or All Set parameters to include desired cell images Increase the squelch setting until less debris is imaged while maintaining cell and bead imaging Advanced Acquisition tab The computer is running Exit other programs other programs that use a lot of processing power INSPIRE Camera is not running Click Run Setup appears to freeze If the camera is already running click Stop to stop the camera and then click Run Setup Imaging is paused Click Resume 106 TROUBLESHOOTING SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS No objects in the current image view mode Toggle image view mode to All to look for objects A script is running If the status indicates the syringes are moving and the Abort Script button is dark a script is running Wait until the script completes or if necess
11. you are ready to acquire experiment data files The sample is loaded into the sample pump Beads and sample are injected into the flow cell to form a single core stream that is hydrodynamically focused in front of the imaging objective The beads are used by the system to keep the autofocus and camera synchronized during the sample run while the objects from the sample are saved to the data file To use the Autosampler for unattended operation see Using the Autosampler on page 33 Refer to the ImageStream Sample Preparation Guide for experimental set up recommendations Use compatable sample solutions from the table below INA SHEATH FLUID ACCEPTABLE PBS PBS Yes PBS Water Yes PBS Surfactant PBS Yes PBS Surfactant Water No Water PBS Yes Water Water Yes Water Surfactant PBS No Water Surfactant Water Yes Cells in PBS run with water sheath will swell OPERATING THE IMAGESTREAM USING INSPIRE 23 SAMPLE ORDER Samples from an experiment are typically run in the following order e Experimental sample with the brightest stains e The rest of the experimental samples Single color DNA dye control NO BF or SSC e 10 bleach to wash out DNA dye Single color fluorescence controls no DNA dye NO BF or SSC LOADING AND RUNNING THE SAMPLE 1 Press Flush Lock Load and load the brightest sample in the experiment that fluoresces with each fluorochrome used
12. 8 00 ASSIST BFTestQualityCh2 3 00 ASSIST BFTestQualityCh3 7 00 ASSIST BFTestQualityCh4 3 00 ASSIST BFTestQualityChS 3 00 The results and limits of the test are shown below of the list when the test is selected INSPIRE SOFTWARE OVERVIEW 69 BF UNIFORMITY TEST Measures the static and temporal uniformity of illumination in all brightfield channels channels 1 through 6 1 12 if the Twelve Channel option is installed Non uniformities in illumination can affect segmentation and the accuracy of photometric absorbance measurements made in the brightfield channel Non uniformities can be caused by misaligned illumination and collection path elements degradation of pixel responsiveness and electronic noise The brightfield uniformity test measures the response from each pixel column the illumination and collection systems are providing a uniform photometric response ES Brightfield Uniformity Test Parameters Num Settings Channel OF Interest Num samples Total Range Pixel Intensity 600 400 253 49 i D 1 1 1 1 1 1 5 3 3 5 4 45 Camera Channel Idle Database Setting Saved Value Result Value ASSIST BFPeakVarition 0 921 020 MMA ASSIST BFPeakVariationMin 0 00 0 00 0 ASSIST BFPeakVariationMax 1 50 1 50 1 ASSIST DarkCurrent0_Gen2_2 22 285156 8SSIST DarkCurrenti_Gen2_2 22 066406 8SSIST DarkCurrent2_Gen2_2 23 039062 8SSIST DarkCurrent3_Gen2_2 24 253906 70 INSPIRE M SOFTWA
13. Calibration Template ist Check the following critical settings 82 INSPIRE SOFTWARE OVERVIEW 9 10 11 12 Collection Filter EDF BF OFF Ex laser 100 mW Ch1 stage setting 256 Ch2 6 512 Percent Beads 10 Diameter 3 microns Cell classification parameters set channels 1 and 3 to area min 25 max 500 Peak Intensity Max 1000 Click Flush Lock Load script to load 50 ul of EDF beads into the sample line To view images click Run Setup and choose the Cells view Imagery in channels 1 and 3 will display the characteristic L shaped spread of the light from the EDF beads as shown Note if single beads as shown are not being characterized in the cell classifier call Amnis support Wait until the flow speed CV is consistently less than 0 2 The events per second should be between 50 200 second Select the EDF Calibration in the ASSIST tab and click Start Calibration Click Start in the Calibration window When the calibration has finished the kernels should look similar to the images shown in the Calibration window below Click SaveDB to save the kernels to the database Close the calibration window INSPIRE SOFTWARE OVERVIEW 83 FOCUS PAN UTILITY The Focus Pan Utility allows the user to define an automatic focus pan to be performed The user can enter the pan range in microns the number of intervals within the pan and the number of samples to be taken per interval The utility can be used in conjun
14. Cells view and are not collected in the sample data file Bead Classifier Allows you to set the classification scheme for beads This is used to eliminate debris that may be misclassified as beads using the auto matic bead detection classifier scheme Data File Folder Displays the folder where the data will be saved Browse Allows you to choose another folder for the data or create a new one File Name Specifies the name of the data file e File names must be 256 or fewer characters in length including the path and file extension e File names cannot contain the following characters lt gt or Sequence Number Appends an extension to the end of the file name The extension is incremented by 1 each time the file is saved Save Bead Image Data When checked all objects classified as beads are automatically saved in a separate file The file s name is appended with beads Save Debris Image Data When checked all objects not classified as cells or beads are automatically saved in a separate file The file s name is appended with debris Path Displays the directory pathway for the data file s Acquisition Cell Count Bead Count Debris Count Displays the current number of objects that are detected by the camera and categorized as cells beads or debris The count is reset when the camera is set to either Run Setup or Run Acquire mode Total Count is the sum o
15. Delta ASSIST FlowCorePositionT est_FocusPos alue 219 16 ASSIST FlowCorePositionT est_FocusDefaultValue 217 50 ASSIST FlowCorePositionTest_LateralDeltaMax 30 00 Calculated Lateral Position Delta The result and the limits for the calibration are shown below the list when the calibration is selected 74 INSPIRE SOFTWARE OVERVIEW FOCUS OFFSET TEST Measures the offset between the focus determined by the AFFS system and location of the peak response of the Image Collection system This test performs a pan through focus while simultaneously collecting SpeedBead focus data from the AFFS system and SpeedBead image data from the image collection system The AFFS data are processed to find the zero crossing point of no defocus and the image data are processed to determine the peak response point of highest spatial resolution Both sets of data are plotted as a function of Z position along the horizontal axis The AFFS zero crossing and image collection system peak response are indicated vertical lines and numerical results are reported to the Focus Offset test tab The difference in microns between these two positions is determined and compared against predetermined limits and stored in the ASSIST database If the MultiMag option is installed a focus offset test will be performed for each magnification INSPIRE SOFTWARE OVERVIEW 75 Focus Offset 40x Test Parameters Num Settings Channel Of Interest Num samples Tot
16. Linear Threshold fo E Linear Threshold 0 C Log Gain 4 C Log Gain textBox Ch3 Channel 9 EE Linear Threshold fo R Linear Threshold 0 C Log Gain 4 C Log Gain textBox Ch4 e Channel 10 Linear Threshold jo Linear Threshold 0 C Log Gain 4 _ C Log Gain textBox Ch5 a Channel 11 Linear Threshold fo Color Linear Threshold 0 C Log Gain 4 E C Log Gain textBox Ch6 Channel 12 Linear Threshold fo Color Linear Threshold 0 C Log Gain 4 _ C Log Gain textBox Saturated Color E Reset All Channels Color INSPIRE SOFTWARE OVERVIEW 99 ACQUISITION TAB The Acquisition tab allows you to customize object detection parameters and manage the naming and saving of data files The Acquisition tab contains the following settings Object Detection Cells to Acquire Allows you to specify the number of objects to acquire and save in a file Squelch Determines the sensitivity of the object detection algorithm to reduce collection of debris The squelch value can be set to any number from 0 to 100 Higher numbers result in less sensitive object detection and fewer debris particles detected Squelch is used for samples with a high object rate 600 objects per second or more due to an excessive amount of debris particles Auto Detect Beads When checked SpeedBeads are automatically classi fied as beads do not appear in the
17. The values on the ASSIST tab are reported as the number of pixels required to bring each channel into perfect spatial registry when the raw image file rif file is processed to generate the compensated image file cif file Values exceeding 0 95 pixel are flagged as errors and will require manual intervention to realign the filter stack assembly The result and the limits for the calibration are shown below the list when the calibration is selected Please note if the 12 channel option is present this calibration will illuminate and calibrate all 12 channels 60 INSPIRE SOFTWARE OVERVIEW DARK CURRENT CALIBRATION Measures and stores 3072 offset values corresponding to pixel columns in the TDI camera Every pixel in a CCD detector is an individual sensor with its own sensitivity characteristics In the absence of any light each pixel emits a signal known as dark current Although the statistical variation of any given pixel over time is less than one count the mean dark current signal generated by any pixel may vary as much as several counts from a different pixel in the array When the ImageStream is measuring very dim signals even one count difference between pixels can be critical Therefore a Dark Current calibration factor is stored for each pixel column This factor is added to or subtracted from each pixel in the rif file during cif creation to normalize detector variation In the cif each pixel is calibrated so that in the
18. absence of light its signal is 30 counts Fe Dark Current Calibration Parameters Num Settings Channel Of Interest Num samples Total Range Mean Intensity N N N N Nh N N Les gt a nm 1 1 1 1 J e 1 1 1 1 1 1 1 1 1 100 200 300 400 500 600 700 800 Camera Pixel Column Idle Database Setting Saved Value Result Value ASSIST DarkCurrent0_Gen2_2 22 121093 8SSIST DarkCurrenti_Gen2_2 22 683593 ASSIST DarkCurrent2_Gen2_2 22 882812 8SSIST DarkCurrent3_Gen2_2 24 589843 85SIST DarkCurrentMax 100 00 Minimum Maximum The Dark Current calibration commands the system to turn off the excitation laser and brightfield illumination The system then measures the mean signal value of each camera column from 1000 rows of data per column The difference between this value and 30 counts is stored for subsequent correction When the camera is operated at different stage settings 32 64 128 256 stages the dark current characteristics of a column of pixels can change Therefore values for all stage settings are stored total of INSPIRE SOFTWARE OVERVIEW 61 3072 values INSPIRE automatically appends the calibration values appropriate for the stage settings used during acquisition to the rif file The values reported on the ASSIST tab indicate the maximum variation detected from all test conditions The result and the limits for the calibration are shown below the list when the calibration is selected If
19. addition run time information from the SpeedBeads is used by the ImageStream to maintain continuous synchronization between the camera and the sample flow rate and to automatically focus on the sample core stream SpeedBead imagery is automatically excluded from image acquisition files Any cell type that can be analyzed on a flow cytometer can also be analyzed on the ImageStream platform The cells analyzed with the ImageStream can be either adherent or non adherent Adherent cells must be placed in suspension via trypsin or similar treatment The cell particle size should be less than 55 microns wide since the field of view on the ImageStream camera is 62 um wide The samples may be passed through a 70 micron mesh filter before running to prevent clogging of the fluidic system for more information see Sample Preparation guidelines The flow cuvette channel dimensions are 250 microns x 875 microns 15 INTRODUCTION 16 INTRODUCTION CHAPTER 3 Operating the ImageStream Using INSPIRE This chapter describes the operation of the ImageStream system using the INSPIRE software Daily operation involves an initial calibration and testing of the system using SpeedBeads and ASSIST followed by sample runs and data acquisition and finally sterilization of the system for use the following day Optimizing instrument setup for sample runs is also described here in detail Fluidics INSPIRE User Interface e Daily Calib
20. consists of an average velocity measurement of approximately 50 SpeedBeads thereby measuring the speed of approximately 5000 SpeedBeads The test computes a running average of all measurements which is listed under results on the pop up window and ensures that no more than 5 of all measurements exceed a 0 15 speed variation This ensures that synchronization is maintained between the imagery and the camera INSPIRE SOFTWARE OVERVIEW 71 to better than a fraction of a pixel Test results are stored in the ASSIST database The results and limits of the test are shown below the list when the test is selected 72 INSPIRE M SOFTWARE OVERVIEW FLOW CORE LATERAL STABILITY TEST Provides a statistical characterization of the stability of the core in the direction lateral to flow The test computes the centroid position of approximately 3000 SpeedBeads During the test a histogram of bead centroid position is plotted in the test window When the test is complete the standard deviation of bead centroid position in pixels is printed in the test window E Flow Core Lateral Stability Test History 3 25 2009 9 13 05 AM Parameters Num Settings Channel Of Interest Num samples Total Range 5 5 2 a E wo 1 1 0 10 X Centroid um Database Setting Saved Value Result Value ASSIST ASSIST LateralPositionStdDevTestValue 5 00 BDD ASSIST LateralPositionStdDevT estMax 15 00 Camera CameraPixelSize 0 50
21. continues to fail Image Quality test failure Verify that the SpeedBeads are running well and that there is no air trapped in the system Run the Purge Bubbles or Sterilize System scripts then repeat ASSIST Call Amnis service if it continues to fail 114 TROUBLESHOOTING Index A Acquisition tab 27 29 100 Advanced Fluidics tab 88 Advanced setup 87 advanced fluidics 88 autofocus 88 camera 95 illumination 98 ASSIST 57 ASSIST Calibrations 59 Autofocus S Curve 79 Brightfield crosstalk coefficient 63 camera synchronization 59 Dark Current 61 Horizontal laser 64 retro 67 side scatter 66 spatial offsets 60 ASSIST Tests 68 BF Intensity Selection 69 BF uniformity 70 Excitation laser power 68 flow core axial stability 71 flow core lateral stability 73 flow core position 74 focus offset 75 image quality ensquared energy test 77 ASSIST Utilities 79 brightfield calibration 81 EDF excitation 82 focus pan 84 PMT focus 85 Auto Detect Beads 100 Autofocus tab 88 B Basic Fluidics 14 Bead Concentration vs Time 90 Brightfield 12 C camera 10 bin mode 95 collection mode 95 gain 95 sensitivity 95 test modes 96 trigger source 95 camera modes frame 96 TDI 96 Camera tab 95 cell detection 50 Channel 51 Cleanser 18 Core Tracking 21 52 D data files 27 Debris Squelching 29 Debubbler 18 Display tab 98 F Flow Speed 22 28 55 56 flow speed 14 92 fluorochrome balancing 24 Frame mode 95 96
22. high Increase or decrease the 785 excitation laser power dim or bright or low for the desired brightness Scatter intensity Instrument is experienc Allow the instrument to warm up by running for changes over ing large temperature 15 minutes time variation Direct a fan toward the back of the instrument to dissipate excess heat Large flow speed varia See Flow speed is not stable on page 109 tion Large variation Large flow speed varia See Flow speed is not stable on page 109 in brightfield tion intensity levels Light source delivering Call Amnis service variable output Brightfield Intensity set before Wait until actual velocity matches the desired intensity level desired flow speed has velocity and then click Set Intensity sets incorrectly been achieved ND filter wheel is dirty Call Amnis service 112 TROUBLESHOOTING SYMPTOM Blue non parsed imagery in all channels Erroneous flu idics sensor reading Instrument will not pass ASSIST POSSIBLE CAUSES This sometimes happens when instrument exits from ASSIST Debris has accumulated on the optical sensor RECOMMENDED SOLUTIONS Toggle between Beads and Cells views Clean sensor with detergent and water Sensor is broken SpeedBeads are not run ning properly Call Amnis service Verify that the core stream is stable and 100 300 beads per second are flowing through the instru
23. is set too low Lock on button The sheath syringe is Load sheath then dual prime empty TROUBLESHOOTING 111 SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS There is a clog or air bub See solution for Air or clog in the sample syringes ble in the system or flow cell One channel Instrument sensitivity is The best instrument setup maximizes the dynamic saturates while not optimized range of scatter and fluorescence signal detection the others do while at the same time avoiding image pixel satura not tion which cannot be compensated in the fluores cence channels Refer to Setting ImageStream Sensitivity for guidance on instrument setup Probing protocol requires Reduce the concentration of the stain that pro better stain balance duces the saturating signal so that all probes can be simultaneously imaged without excessive satura tion Cross contami DNA dye from previous DNA dyes must be thoroughly flushed from the nation from sample is labeling current sample lines syringe and flow cell to prevent resid previous sam sample ual dye from labeling subsequent samples that do ples not contain the same DNA dye Run the Flush Lock and Load script with 10 bleach to remove all traces of the DNA dye Cells from the previous This suggests a minor clog Run the Sterilizer sample are appearing in script to remove all traces of the previous sample current sample Scatter is too Laser power set too
24. laser power INSPIRE SOFTWARE OVERVIEW 51 OBJECTIVE STAGE POSITIONING Controls for moving the imaging objective lens Core Tracking microns Relative fio A D Current 0 0 Reset Focus Tracking microns Relative f gt Current 13 Reset Auto tracking Q e Core Tracking Provides controls that move the objective in the x axis so that object images are collected in the center of the camera channels All values are expressed in microns Relative You can enter the distance that the objective stage will be moved in the x axis when the right or left arrows are clicked This positions the objective to center images laterally within the objective s field of view Current Records the current position of the objective stage in the x axis Reset Returns the objective to the original x axis position specified in the INSPIRE template e Focus Tracking Provides controls that move the objective in the z axis to achieve optimal focus on objects in the core stream All values are expressed in microns Relative You can enter the distance that the objective stage will be moved in the z axis when the right or left arrows are clicked This positions the objective to achieve optimal focus on the core stream Current Records the current position of the objective stage in the z axis Reset Returns the objective to the original z axis position specified in the INSPIRE template Auto tracki
25. ment at 60 mm sec Incorrect template loaded Load the Default template Camera Synchronization calibration failed causing all other calibrations and tests to fail If Camera Synchronization fails manually enter the last known good value 38 5 and run ASSIST again Note that if this calibration fails most of the other calibrations will fail as well Spatial Offsets calibration failure Verify the camera synchronization value and run Spatial Offsets calibration again If it will not pass the filter stack is misaligned call Amnis service Dark Current calibration failure Make sure the 488 excitation laser is off and bright field is blocked If it fails the Dark Current calibra tion a second time call Amnis service Brightfield XTalk Coef ficicient calibration fail ure Run the calibration a second time and if it fails call Amnis service AutoFocus S Curve cali bration failure Verify the camera synchronization value and re run the Laser Alignment calibration until it passes If it continues to fail call Amnis service 405 488 658 785 Laser calibration failure Verify the camera synchronization value and re run the Laser Alignment calibration until it passes If it continues to fail call Amnis service Side Scatter calibration failure Run the calibration a second time and if it fails call Amnis service Retro calibration failure Run the calibration a second time a
26. or within cells or between cells in cell conjugates INTRODUCTION 9 The Amnis ImageStream system includes the ImageStream multispectral imaging flow cytometer and the INSPIRE and IDEAS software applications The INSPIRE software is integrated with the ImageStream and is used to run the instrument INSPIRE also provides tools for configuring the ImageStream defining cell parameters and collecting data files for image analysis The IDEAS software is used for spectral compensation image analysis as well as statistical analysis of the images acquired by the ImageStream multispectral imaging flow cytometer TECHNOLOGY OVERVIEW 10 The ImageStream acquires up to twelve images simultaneously of each cell or object including brightfield scatter and multiple fluorescent images at rates of up to 1000 objects per second The time delay integration TDI detection technology used by the ImageStream CCD camera allows up to 1000 times more signal to be acquired from cells in flow than from conventional frame imaging approaches Velocity detection and autofocus systems maintain proper camera synchronization and focus during the process of image acquisition The following diagram illustrates how the ImageStream works detector dichroic filter stack g velocity detector A om CO autofocus cells in flow p J f J brightfield illuminator Hydrodynamically focused cells are trans illuminated by a
27. pumps The sheath pump helps to control the speed of the core stream and the size of the core stream diameter The flush pump is used to clean and flush the system 18 OPERATING THE IMAGESTREAM UsiNnG INSPIRE INSPIRE USER INTERFACE The INSPIRE user interface displays imagery and acquisition plots and the controls for setting up and acquiring image data Below is a brief description of the steps for running the ImageStream The rest of this chapter describes these steps in detail Launch INSPIRE 11 Open Template if appl cabl 1 2 Load Default Template 12 Set the Laser Power 3 Click Initialize Fluidics 13 Select EDF collection if desired 4 select ALL in Image Gallery 14 Set Cell Classification criteria 5 Press Run SetUp 15 File Name Destination Folder 6 Adjust Core Tracking Sequence Number of Events 7 Set Brightfield BF Click Set Intensity 16 Click Run Acquire 8 Monitor Flow Speed CV 17 Click FLL to Load next sample 9 In the ASSIST tab click Start All 20 If finished for the day choose Sterilize System 10 Flush Lock and Load FLL to Load sample OPERATING THE IMAGESTREAM USING INSPIRE 19 DAILY CALIBRATION AND TESTING TURNING ON THE IMAGESTREAM X This section describes how to prepare the ImageStream for use The ImageStream is usually left on with INSPIRE launched but the following instructions also describe how to turn the ImageStream on if the power
28. signal is used in a control loop to make minute corrections in the position of the objective lens to maintain accurate focus OBJECT DETECTION In TDI mode the CCD camera collects a continuous stream of imagery corresponding to the continuous stream of core fluid passing through the detection region Objects within the core stream appear as discrete events often with large blank image areas between them The object detection algorithm retains images containing object events and discards the blank imagery between them To do this the algorithm sums the magnitudes of intensity gradients in the imagery and signals a detection event whenever it finds an image region where the summed gradients are significantly larger than those of the surrounding background Only images that contain objects detected by the algorithm appear in the acquired data file HYDRODYNAMIC FOCUSING 14 Hydrodynamic focusing is used to position the beads and cells within a core stream in a consistent place within the flow cell This is achieved by running the microstepper driven sheath sample and SpeedBead pumps at differential speeds SpeedBeads and cells are introduced together above the flow cell in a ratio determined by their relative pump speeds In the flow cell the mixture is injected into the center of a sheath stream that is running at a constant velocity greater than that of the beads and cells to form a core stream To enable higher cell imaging throughput
29. that will achieve the desired core stream Diameter Percentage Beads and Desired speed e Lock On button Speeds up or slows down the fluidics to achieve the Desired speed e Stop Stops the fluidics INSPIRE SOFTWARE OVERVIEW 53 OPTIONS Controls for optional magnification binning and EDF Magnification Binning C 20x 40x 60x N E 2 Collection Filter e Collection Filter Allows control of the collection filter for different laser illuminations Open No collection path filter ND 1 2 3 Neutral density filters used during ASSIST EDF1 Wave front coded element used for extended depth of field Block Blocking element used during ASSIST 54 INSPIRE SOFTWARE OVERVIEW STATUS The Status area contains information regarding the core stream fluid levels and script processes for monitoring the system Status Switch Sheath Pump Script Complete Script Log Fluid Levels Syringe Volumes Sample Portal Sheath RS ccc D J URS 24500 00 uL 424 69 uL Down Sample Flush D 0 00 uL 16421 71 uL Vial Loaded Flow Speed Amplitude Flow Speed CY 0 4 3 0 3 TT 2 02 wo S01 B mars AP An 0 1 I I 0 4 1 I I 0 20 40 60 72 74889 75000 75089 Core Velocity mm sec Observations Status area Script Running Indicator light that is bright green when a fluidic script is running Text Box Updates the progress of the current script and provides a fluidic script
30. the ImageStream incorporates a unique wide aspect flow cell with a flow channel that is 3 5 times wider in the image plane than in the focus dimension As a result of hydrodynamic focusing isolated cells and beads form into a broad flat flow or ribbon within the core stream The dimensions of the core stream are increased by increasing the sample and bead pump velocities and decreased by decreasing the sample and bead pump velocities The core stream flow speed is controlled by proportionally changing the speed of all three pumps INTRODUCTION SPEEDBEADS AND CELL SAMPLES SpeedBeads and sample are introduced above the flow cell and injected together through the canula into a flowing column of sheath to form a core stream that is hydrodynamically focused in front of the objective in the flow cell cuvette Sample and SpeedBeads are drawn into the system with separate glass syringe pumps Samples are loaded from a 1 5 ml capacity microcentrifuge tube placed on the sample intake port The SpeedBead reagent comes in 15 ml conical tubes and is loaded onto the bead port behind the fluidics door on the front of the instrument SPEEDBEADS CELLS The SpeedBead reagent is integral to the operation of the ImageStream system and is run with each sample SpeedBeads are used to test and calibrate the instruments illumination optical camera and fluidic systems at the beginning of each day for more information see Daily Calibration and Testing In
31. the plate definition At a minimum each well requires an Output File Path Max Acquisition Time Cell Count and Template File in order to be considered defined Other param eters can optionally be added to the definition in the next step 5 Choose the parameters you would like to use e Click Add Remove Well Parameters to choose the parameters you want to report for the wells Add or Remove Columns C Standard Parameters C Custom Parameters H O Display Parameters Fluidics Parameters l C Illumination Parameters C Imaging Parameters Enter custom column name here Add Custom Column OK Cancel There are several categories of parameters that may be chosen as a group or indi vidually See the list of parameter below Well Parameters on page 38 Check or uncheck the desired parameters The user can also define custom parameters 34 OPERATING THE IMAGESTREAM USING INSPIRE Expand the category to see the individual parameters To delete a custom parame ter select it and use the delete key Click OK when done To include a parameter in the file name click in the box below the column heading make sure it says yes e Columns can be re ordered by click drag e Click OK when finished adding or removing parameters 6 Define the wells Select wells to define by clicking a individually orCtrl click shift click for multi select b rows or columns c color d the
32. which the focus error varies linearly with the amount of defocus This calibration measures and stores the slope of the linear region of this curve focus error score microns The Autofocus system in the ImageStream is capable of detecting focus variations as small as 50 nm To maintain diffraction limited performance the Autofocus system measures and corrects any deviations in focus When the Autofocus system detects a focus error it commands a stage to move the objective lens to correct the error The actual move distance is the quotient of the autofocus signal error and the slope of the response curve Autofocus S Curve Calibration Parameters Num Settings Channel Of Interest Num samples Total Range 2 5 te on a 5 ts res 1 1 0 2 Focus Position um Idle Database Setting Saved Value Result Value ASSIST SCurveSlope 0 23 ASSIST SCurveSlopeMin 0 12 8SSIST SCurveSlopeMax 0 28 INSPIRE M SOFTWARE OVERVIEW 79 The Autofocus calibration moves the stage approximately 5 microns out of ideal focus and collects focus error scores from 100 SpeedBeads The stage then moves one micron toward focus and collects focus error scores again The process is repeated until the stage reaches a position 5 microns beyond best focus The data are then fit to a 3rd order polynomial and the slope of the curve at the inflection point is determined This value determines the signal error corresponding to a given focus error th
33. 3010 WE CDASFITC iet Gars com CDASF TNF Ficom 100p9 60min a nat EDATE Ble c Documents and Setings amris Desktop ISX O ata 043010 101 WB MONO NFkB TNF 15 5000 043010 WB CD4SFITC it Garris com CD4SF TNF PE ctl 1009 60min a3 Hoag DATE El C Documents and Setting tamis Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 049010 WB CD4SFITC ist Gars com CD4SF TNF AFBA7 cut 1p9 60min m aout DATE Bide C Docurents and Settings smris Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 043010 WE CDASFITC iet onris com CD4SF TNF 100 60min A5 posg DATE El C Documents and Settings amis Desktop SX Data 043010X101 WB MONO NFkB TNF 15 5000 043010 WB CD4SFITC ist arr com COASF TNF 1069 60min A6 os DAT C Documents and Setting amis Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 043010 WB CDASTC it Ganiscom CD45F TNF DAPI 19 60min aT aom EDATE El C Documents and Setinge amris Deaktop IS Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WE CDASFITC iat anwis com CD45F TNF 10089 60min 48 aosa DAT C Documents and Setings amnis Deshiop USX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 W8 CO4SFITC it anwia com CDASF TNF Unix 60min BI Boys 0 C Documents and Settings arres Desktop ISX Data 043010X101 WB MONO NFKB TNF 15 5000 043010 WB CDASFITC iet arris com CD4SF TNF A 100pq 15min B2 cam HOATE fled C Documents and Setings amnis Desktop ISX D ata 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CDASFITC ist amri
34. 59 18 r Collection Filter gt O open El Status Switch Sheath Pump Script Complete Script Log Fluid Levels Syringe Volumes Sample Portal Se SN lt lt DE g KS 24500 00 uL 424 69 uL Don Sample Flush M ut aie Vial Loaded Flow Speed Amplitude Flow Speed CY 04 5 03 3 2 02 wo 8 01 uw RNA or 1 D 1 1 0 1 I 0 20 40 60 72 74889 75000 75089 Core Velocity mm sec Observations INSPIRE SOFTWARE OVERVIEW 49 DATA ACQUISITION The following two areas of the Setup tab pertain to ImageStream data file acquisition Acquisition CelstoAcqure S000 Data File Filename 3 Sequence 1 Save Debris e Acquisition box Cells to Acquire Allows you to specify the number of objects to acquire and store in a file Save Debris checkbox When checked all objects not classified as cells or beads are automatically saved to a separate file This file s name is appended with debris Data File You can specify the data file name and browse for a folder in which to save the data Sequence This number is appended to each data file name and ascends with each file acquired e Acquisition Status box Acqusition Status CelCount 0 Cals Sec 514 Total Sec 514 Elapsed 0 Cell Count Displays the current number of cells detected by the camera Counting is reset when the camera is set to Run Setup or Run Acquire mode Cells Sec Displays the cell
35. ACTURER Amnis Corporation 2505 Third Avenue Suite 210 Seattle WA 98121 Phone 206 576 6865 MODEL NUMBER ImageStream X REPORT AMNI0006 and F2LQ3326 02S DIRECTIVES EMC Directive 2004 108 EC amp Low Voltage Directive 2006 95 EC STANDARDS Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements EN 61326 1 2006 edition Industrial Scientific and Medical Equipment Radio Frequency Disturbance Characteristics Limits and Methods of Measurement CISPR 11 2009 edition Information Technology Equipment Radio Disturbance Characteristics Limits and Methods of Measurement EN 55022 1998 CISPR 22 1997 edition Electromagnetic Compatibility EMC Part 3 2 Limits Limits for Harmonic and Emissions Equipment Input Current lt 16 A per phase IEC 61000 3 2 2009 edition EMC Part 3 3 Limits Limitations of voltage changes voltage fluctuations and flicker in public low voltage supply systems for equipment with rated current lt 16 A per phase and not subject to conditional connections IEC 61000 3 3 1995 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 2 Electrostatic Discharge Immunity Test EN 61000 4 2 2008 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 3 Radiated Radio Frequency Electromagnetic Field Immunity Test EN 610004 3 2008 edition Electromagnetic Compat
36. Contaminated sheath obstructions air or improper pump function may broaden the core which can reduce focus consistency and increase variation in intensity measurements This flow core lateral stability ensures the core is operating as designed with minimal variation Failure to pass this test is indicative of at least one of the issues listed above The result and the limits for the calibration are shown below the list when the calibration is selected INSPIRE SOFTWARE OVERVIEW 73 FLOW CORE POSITION TEST Measures the position of the core relative to its ideal position within the flow cuvette The ImageStream uses sheath flow to hydrodynamically focus objects within a precise region in the cuvette Improper sheath solution protein buildup micro bubbles and other factors can alter the position of the core within the cuvette If this occurs the photometric and morphological measurement repeatability may degrade This test measures the current core position and compares it to the ideal location of the core as determined in the manufacturing process The deviation from the ideal position is reported in microns and stored in the ASSIST database i Flow Core Position Test Parameters Num Settings 1 Channel Of Interest Num samples 1 Td Total Range as TR AC RE NE AR A ELA EL Restoring Instrument Settings Database Setting Saved Value Result Value ASSIST FlowCorePositionT est_FocusD eltaMax 15 00 Calculated Focus Position
37. Definition def that assigns instrument settings to wells names to the sample output files and parameters to include in the plate report see procedure below Add 75vl samples to the 96 well plate cover with Sigma Aldrich X Pierce Film XP 100 Cat 2722502 and load the plate into the autosampler Run the plate see procedure below Access to AutoSampler operations is found under the AutoSampler menu From this menu you may Create a plate definition Atosencier e Run a plate Define Plate Run Piste Run a single well from a plate ser Run Singe Well Extend or Retract the nest Extend Nest Retract Nest 33 OPERATING THE IMAGESTREAMX UsinG INSPIRE TO BEGIN 1 Choose Define Plate from the Autosampler menu to open the Well Plate Defini tion window Swell Plate Definition O Stort E Add Remove Well Parameters Output Max Acquistion Call FilePath Timefnin Count Include in filename mo no ro Plate Definition Name a Customize Well Plate _ Apply to selected Select ty Colo i A M Select Defined Delete Selected Welle mf efe eeehe eol 189555555555 pau ro Bomi 802 eget noate e 805p CN required lor complete well definition 2 Begin to create a new definition or you may browse for a previously saved defini tion to edit by clicking on the folder icon Name
38. F Open C ND2 C EDF2 C ND3 C EDF3 INSPIRE M SOFTWARE OVERVIEW 97 ILLUMINATION TAB The Illumination tab is used to control the brightfield Leds and fluorescence excitation laser settings These controls are to be used under the guidance of Amnis service personnel only DISPLAY TAB The Display tab allows you to customize the image display characteristics in each camera channel in the Imaging area of INSPIRE The display settings do not affect the raw data that is collected The following image shows typical settings The Display tab contains the following settings for each channel Linear or Log Sets the scaling of the image display look up table Threshold Sets the background intensity level e Gain Sets the amplification level e Color Determines the display color that is used for the channel The color is used both for the images and for the dots in the acquisition plots for the chan nel Saturated Color Determines the color used to display saturated camera pix els that is pixels with the maximum intensity 1023 arbitrary units Reset All Channels Color Turns all channel colors to white 98 INSPIRE SOFTWARE OVERVIEW Setup ASSIST Advanced Setup Advanced Fluidics Autofocus Flow Speed Camera Illumination Display Acquisition M Color Transformations Chi Channel 7 Linear Threshold fo 2 Linear Threshold 0 Color C Log Gain 4 C Log Gain textBox Ch2 Channel 8
39. Frequency Spectrum 90 FSP 88 G gain camera 95 graphs Autofocus Stage Position 90 Bead Concentration vs Time 90 focus 90 Frequency Spectrum 90 Time Series 90 H Hydrodynamic Focusing 14 l Illumination tab 98 ImageStream shutting down 30 starting 20 Imaging Area 40 Imaging Tools 40 L laser 10 Laser Excitation 12 loading beads 21 o Object Detection 14 optics 12 optimizing settings 17 P password 20 PMT settings 85 R reagents 18 S Safety Biological Safety 8 Electrical Safety 4 Laser Safety 5 Sample 15 sensitivity camera 87 95 sheath bottle 18 sheath fluid 18 SpeedBeads 15 Sterilize System 41 115 116 Sterilizer 18 Synchronize Channels 95 T TDI mode 14 95 96 Time Series 90 tracking core 52 trigger source 95 U user name 20 Vv valves 88 View Raw Frames 95 W waste 4 8 fluid 18
40. INSPIRE IMAG ESTREAM SYSTEM SOFTWARE USER S MANUAL VERSION 4 0 JULY 2010 Amnis Corporation 2505 Third Avenue Suite 210 Seattle WA 98121 800 730 7147 www amnis com Patents and Trademarks Amnis Corporation s technologies and products are protected under one or more of the following U S patents 6211955 6249341 6256096 6473176 6507391 6532061 6563583 6580504 6583865 6608680 6608682 6618140 6671044 6707551 6763149 6778263 6875973 6906792 6934408 6947128 6947136 6975400 7006710 7009651 7057732 7079708 7087877 7190832 7221457 7286719 7315357 7450229 7522758 7567695 Additional U S and corresponding foreign patent applications are pending Amnis the Amnis logo ImageStream INSPIRE IDEAS SpeedBead FISHIS are registered or pending U S trademarks of Amnis Corporation All other trademarks are acknowledged Disclaimers The screen shots presented in this manual may vary in appearance from those on your computer depending on your display settings The Amnis ImageStream cell analysis system is for research use only and not for use in diagnostic procedures Technical Assistance Amnis Corporation 2505 Third Avenue Suite 210 Seattle WA 98121 Phone 206 374 7000 Toll free 800 730 7147 Wwww amnis com TABLE OF CONTENTS CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 INFORMATION AND SAFETY General Information and Safety Declarat
41. Intensity Selection Test fails Run ASSIST daily to optimize the performance of the ImageStream To run ASSIST calibrations and tests 1 Click Start All Calibrations and Tests to run all standard calibrations and tests 2 Torun one calibration or test click on an individual calibration or test and click Run 3 To stop a calibration or test click Stop or Stop All if Start All was chosen A calibration or test will be flagged red if it fails If a calibration or test fails run that calibration or test individually and if it fails again call or email Amnis service INSPIRE SOFTWARE OVERVIEW 57 Setup ASSIST Advanced Setup Calibrations Calibration Last Run Time Spatial Offsets Calibration Camera Synchronization 40 Calibration 10 29 2009 8 57 49 AM 10 28 2009 8 46 42 PM Dark Current Calibration Brightfield XT alk Coefficient Calibration 10 28 2009 8 46 50 PM 10 28 2009 8 47 18 PM Autofocus Curve Calibration 10 28 2009 8 47 41 PM 405 Horizontal Laser Calibration 10 28 2009 8 48 25 PM 488 Horizontal Laser Calibration 10 28 2009 8 49 21 PM 658 Horizontal Laser Calibration 10 28 2009 8 50 11 PM 785 Horizontal Laser Calibration 10 28 2009 8 51 01 PM Side Scatter 40x Calibration 10 28 2009 8 51 28 PM Retro Calibration 10 28 2009 8 52 27 PM Tests Start All Ca Last Run Time Ibrations 405nm Laser Power T
42. O Core Diameter M Color _ Core Velocity Output File Path Illumination Parameters M Max Acquisition Time min Q BF Intensity M Cell Count BF Channel Template File O 405 SSC Blocker M Error Notification Email Fes Power O Comments 488 Power D Dose 561 SSC Blocker O Time 0561 Power C Custom Parameters O 592 SSC Blocker oc E60 SSC Bk CO Cel er O Treatment 560 Power O Stain O 785 Power Display Parameters 830 Power choi Imaging Parameters C cho2 C Aperture O chos C uto Bead Detection C chos L Binning o chos O Cross Camera Reference Channel C chos C INSPIRE BF Gains Comected O Cho 1 Magnification c chos Save Bead Fie c chos C Save Debris File C Ch10 O Squeich Och Oichi2 38 OPERATING THE IMAGESTREAM USING INSPIRE CHAPTER 4 INSPIRE Software Overview The INSPIRE user screen provides the interface for operating the ImageStream INSPIRE provides basic and advanced control over the fluidic optical illumination detection autofocus flow speed and object classification systems in order to optimize the acquisition of image data into a file INSPIRE USER INTERFACE INSPIRE SOFTWARE OVERVIEW 39 The left side of the INSPIRE window contains e Menu Bar Access the instrument setup and control pull down menus INSPIRE for the ImageStream File Instrument Sample Beads AutoSampler Help lt e Action Buttons Run Acquire Stop Flush Lock Load Retur
43. OFTWARE OVERVIEW es 488 Horizontal Laser Calibration Parameters Num Settings Channel Of Interest Num samples Total Range 2 kA E 2 Tl 1 1 1 20 0 20 Horizontal Laser Position um Idle Database Setting Saved Value Result Value ASSIST ExcHorizontalLaserPosition_488 9 22 2 ASSIST ExcPowerT est_LaserPower_488 100 00 The result for the calibration are shown below the list when the calibration is selected INSPIRE SOFTWARE OVERVIEW 65 SIDE SCATTER CALIBRATION The purpose of this calibration is to set the power of the 785nm laser The calibration routine consists of measuring SpeedBead intensities at a predefined power setting and then actively adjusting the power to achieve 7200 counts of light per bead This calibration ensures a consistent intensity for subsequent ASSIST testing and also ensures a consistent starting position for scatter laser power when analyzing cells ES Side Scatter 40x Calibration Parameters Num Settings Channel Of Interest Num samples E Total Range vs Intensity 2 1 1 6 7 Power mw Database Setting Saved Value Result Value ASSIST SideScatterLaserlntensityMin 6 400 00 ASSIST SideScatterLaserlntensityMax 8 000 00 ASSIST SideScatterLaserlntensityT arget 7 200 00 ASSIST SideScatterCalibintensity 69329 VA ASSIST SideS catterCalibPower 1 92 66 INSPIRE SOFTWARE OVERVIEW RETRO CALIBRATION The ImageStream use
44. OM ns Mae EEA ET A re 4 nein te 8 10 11 15 17 17 19 39 40 43 45 46 iii CHAPTER 5 iv Setup Tab ci aaae r els a oh sade E E E E T 49 ASSIST TaD 44 bid sagdus abi peatata e iea uie edit 57 Advanced Setup Tabra miah LE LR ns aeS AA A ne sa Dr nie 87 TROUBLESHOOTING 103 CHAPTER 1 Information and Safety This section covers safety information for operating the Amnis ImageStream multispectral imaging flow cytometer Anyone who operates the ImageStream should be familiar with this safety information Keep this information readily available for all users The safety information consists of the following areas e General Information and Safety e Explanation of Symbols e Electrical Safety Laser Safety e Biological Safety e Spare Parts List GENERAL INFORMATION AND SAFETY The ImageStream imaging flow cytometer is manufactured by Amnis Corporation and has a rated voltage of 100 240 VAC a rated frequency of 50 60 Hz and a rated current of 3 A The years of construction were 2004 2009 and the product contains CE Marking Environmental conditions This instrument was designed for indoor use at an altitude of less than 2000 m at a temperature from 5 C through 40 C and at a maximum relative humidity of 80 for temperatures up to 31 C with the maximum relative humidity decreasing linearly to 50 at 40 C The main s supply may not fluctuate more t
45. RE OVERVIEW FLOW CORE AXIAL STABILITY TEST Measures the stability of the core stream velocity over time Measures the variation in the speed of the core stream as a percentage of the mean sample speed The ImageStream is designed to automatically sterilize cleanse and purge air from its fluidics systems after every day of operation Improper sterilization contaminants partially clogged fluidic lines air bubbles or non homogenous sheath solution can lead to excessive sample speed variation Although the ImageStream very accurately measures the sample speed to synchronize camera line rate with cell movement on the detector excessive speed variation can lead to small amounts of desynchronization The flow core axial stability test verifies that the fluidic system is operating within normal limits thereby providing the collection system with hydrodynamically focused objects traveling at a consistent speed for proper image synchronization E Flow Core Axial Stability Test Parameters Num Settings Channel OF Interest Num samples Total Range 0 10471 0 08 0 06 0 04 0 02 0 00077178 1 1 1 1 1 1 1 1 D 1 0 10 20 30 40 50 60 70 80 30 100 Sample Number Restoring Instrument Settings Database Setting Saved Value Result Value ASSIST 4SSIST FlowSpeedCV 0 03 ASSIST FlowSpeedCVMax 0 15 ASSIST FlowSpeedCV_PercentOverMax 0 10 The flow core axial stability test plots 100 flow speed sample intervals each of which
46. ageStream is a Class 1 laser device and complies with the U S FDA Center for Devices and Radiological Health 21 CFR Chapter 1 Subchapter J No laser radiation is accessible to the user during normal instrument operation When the hood is opened interlocks on the hood turn the lasers off The ImageStream may have the following lasers TABLE 2 WAVELENGTH MAXIMUM POWER 370 380 nm 30 mW 400 413 nm 150 mW 486 490 nm 100 mW or 500 mW high power option 558 562 nm 200 mW 592 593 nm 300 mW 652 664 nm 130 mW 775 800 nm 80 mW 815 840 nm 180 mW The following laser warning label appears on the inside surface of the hood DANGER Visible and invisible laser radiation when open and interlocks defeated AVOID EYE OR SKIN INFORMATION AND SAFETY 5 The following laser warning label appears on the interior side panels near release mechanisms and next to hood latch release Caution Using controls making adjustments or performing procedures other than those specified in this manual may result in hazardous exposure to laser radiation 6 INFORMATION AND SAFETY S CURIT LASER L ImageStream c est une appareil au laser Classe I qui se conforme U S FDA Center for Devices and Radiological Health 21 CFR Chapitre 1 subchapitre J Aucune radiations laser sont accessible a l utilsateur pendant le fonctionnement normal Quand le capot est ouvert les enclenchements eteindents les l
47. al Range 0 6 05 0 4 03 Ensquared Energy Ratio 0 2 4 0 060156 MM 7 6 0 2 Focus Position um 1 2374 ile 2 5 fa oa a H 3 ts re 1 1 0 2 Focus Position um Idle Database Setting Saved Value Result Value ASSIST FocusOffsetTest Value 0 60 80 ASSIST FocusOffsetT est_LimitMin 0 50 ASSIST FocusOffsetT est_LimitMax 2 50 ASSIST SideScatterCalibPower 1 92 The results and limits of the test are shown below the list when the test is selected 76 INSPIRE SOFTWARE OVERVIEW IMAGE QUALITY ENSQUARED ENERGY TEST Measures the ability of the optical system to resolve fine details in the image using ensquared energy ratio The optics term ensquared energy refers to a measure of concentration of energy in an optical image when quantifying image sharpness for digital imaging cameras using pixels The ensquared energy ratio is one of several parameters often used in the design of high resolution optical systems to characterize their performance In this ASSIST test the ensquared energy ratio of a 3x3 pixel array centered within an 11x11 pixel array is determined and compared against predetermined limits The test is designed to measure the optical quality of the image independent of focus lateral core stability and axial core stability During the test approximately 5000 SpeedBead images are collected over a range of focus positions The imagery is analyzed during collection by computing th
48. am velocity Set the desired speed to 23 mm sec and click the is set too low Lock on button The sheath syringe is Load sheath then dual prime empty There is a clog or air bub See solution for Air or clog in the sample syringes ble in the system or flow cell One channel Instrument sensitivity is The best instrument setup maximizes the dynamic saturates while not optimized range of scatter and fluorescence signal detection the others do while at the same time avoiding image pixel satura not tion which cannot be compensated in the fluores cence channels Refer to Setting ImageStream Sensitivity for guidance on instrument setup Probing protocol requires Reduce the concentration of the stain that pro better stain balance duces the saturating signal so that all probes can be simultaneously imaged without excessive satura tion Everything is Image display settings are Decrease the image display gain and change to lin too bright or set too high ear in the appropriate camera channel images are red or flat in appearance Instrument sensitivity is Decrease the excitation laser power minimum is 10 set too high mW Decrease the camera stage setting minimum is 32 rows to decrease camera sensitivity in the fluores cent channel Refer to Setting ImageStream Sen sitivity guidelines Set the brightfield intensity to 800 counts The core stream velocity Set the desired speed to 23 mm sec and click the
49. annel e Acquisition Provides advanced control over object detection and bead classi fication parameters and allows you to define data file characteristics e Configuration This tab is for Amnis service only and is password protected INSPIRE SOFTWARE OVERVIEW 87 ADVANCED FLUIDICS TAB The Advanced fluidics tab enables control of all fluidic components including filling and emptying pumps and moving valves These controls are to be used under the guidance of Amnis service personnel only AUTOFOCUS TAB The ImageStream uses hydrodynamic focusing to position the core stream which contains beads and cells in a consistent place within the flow cell in front of the imaging objective The imaging objective is mounted on a movable stage that allows the instrument to track back to front z axis and lateral x axis deviations in core stream position in order to keep object images in focus and laterally centered on the camera X axis tracking is called Core Tracking and z axis tracking is called Focus Tracking The Autofocus tab provides control over the Core and Focus Tracking systems as well as real time graphs pertaining to the performance of the Autofocus system The features that allows control of the Focus Tracking and Core Tracking systems with the Autofocus tab are described below Focus Tracking is accomplished by moving the objective in the z axis relative to the core stream The ImageStream uses SpeedBead IR laser side sca
50. ary click Abort Script to prematurely stop the operation Images appear streaked The INSPIRE applica tion has crashed Core stream is moving too fast for the camera Open the Windows Task Manager by simulta neously pressing lt Ctrl Alt Del gt Click the Applications tab If the INSPIRE status is Not Responding select the INSPIRE task and click End Now Restart the INSPIRE application by double clicking the INSPIRE for the Imag eStream icon on the desktop If the program restarts make sure the lasers and brightfield lamp are turned on and then re establish the core stream If the application does not start use the Windows Task Manager to end the INSPIRE task again Shut the ImageStream computer down from the Start menu Then turn on the instrument as described If a crash occurs during the day a complete shutdown is recommended at the end of the day prior to run ning the sterilize script Briefly open the bubble vent Advanced Flow Speed tab to drop the actual flow speed to 60 mm sec or lower Make sure the desired velocity setting is set to 60 mm sec or lower Click Lock on to acquire the desired core stream velocity Camera synchronization setting is incorrect The camera synch value ASSIST tab should not change significantly from day to day If the value is radically different 2 0 re calibrate the camera synchronization using ASSIST or manually set it to a value that has pre
51. asers ImageStream peut avoir les lasers suivants TABLE 3 eee LA PUISSANCE MAXIMALE 370 380 nm 30 mW 400 413 nm 150 mW 486 490 nm 100 mW ou 500mW 558 562 nm 200 mW 592 593 nm 300 mW 652 664 nm 130 mW 775 800 nm 80 mW 815 840 nm 180 mW Les etiquettes d avertissement suivantes sont place s dans V interior du capot DANGER Visible et invisible radiations laser quand ouverte et es enclechements sont defaites Exposition dangereuse au rayonnement direct ou diffus pour losl et la peau Laser Classe 4 dans l appareil laser Classe 1 Les etiquettes d avertissement suivantes sont place s dans L Int rieur de panneaux lat raux pr s de m canismes de lib ration et c t du loquet de fermeture de capot Avertissement L utilisation des commandes ou les rendement des procedures autres que celle precise s aux presentes peuvent provoquer une radioexposition dangereuse 7 INFORMATION AND SAFETY BIOLOGICAL SAFETY Biohazards The Image Stream is rated at BSL1 Do not load or flush samples containing infectious agents without first exposing the sample to inactivating conditions It is recommended that samples be fixed in 2 paraformaldehyde for at least 10 minutes before running the samples on the ImageStream The use containment and disposal of biologically hazardous materials are required to be in accordance with Personnel Protective Equipment Directive 93 95 E and are t
52. ata file You can set upper and lower limits for the following features in any of the six channel images For more information about setting cell classification parameters see Set Cell Classification criteria on page 25 48 INSPIRE SOFTWARE OVERVIEW SETUP TAB The Setup tab provides the user interface for routine operation of the ImageStream On this tab you can monitor and control instrument setup the sample run conditions and data acquisition The Setup tab covers the main features that you must set up data acquisition illumination objective stage position and fluidics These features are described in more detail below Setup ASSIST Advanced Setup Acquisition Cells to Acquire 1000 Data File 102iurkatcells casses 192 Acquisition Status Cell Count a Sequence fi I Save Debris Total Sec J 1725 Elapsed J 0 Brightfield r Excitation Lasers r Core Tracking microns are On Off Intensity mw Relative 110 405nm B 30 cunent 0 0 Reset Intensity 732 ici Ji 488nm C B 100 0 Channel fi z m Focus Tracking microns esen faoz Peetivefi DI Set Intensity 785 nm D a Current F3 Reset Fluidics 7 Auto tracking Q Diameter microns E o Percent Beads fico ASSIST Status 10 29 2009 9 39 03 AM Core Velocity mm sec Tee Magnification inning Desired Jeo z si ax CE None X Actual
53. ation helps ensure crisp image collection E Camera Synchronization Calibration Parametere Hum Settings COS Channa Of Interest Num samples Gn n r Tota Range 1 1677 11 1 as DHL 07 061029 n d 31 w5 33 5 40 ae Camera Sync Sync Gred Metic Sync Gred Mettic 1 1002 HT se 1 y 1 1 38 1 5 3 5 40 ane Camera Sync LCLLLLEECLLLLE CELL EEELEEEECELELCECCCEEECECEC Ide Database Setting Saved Vale Renit Value ASSIST CameraSyncStat 3425 CosersMagn 3500 CameraMagMax 4200 ASSIST ComersMag niu 14 Camere 2nd Camera As shown in the figure above the camera synch calibration measures SpeedBead ellipticity at numerous discrete camera synch settings and plots the camera synch setting horizontal axis versus the ellipticity vertical axis It then generates the best fit curve for a 4th order polynomial through the data and determines the horizontal location camera synch of the peak of the curve The peak occurs where the SpeedBeads appear round This setting is then stored and used for all subsequent image acquisitions The result and the limits for the calibration are shown below the list when the calibration is selected Please note that Camera Synchronization Calibrations will be done for each magnification present in the system INSPIRE SOFTWARE OVERVIEW 59 SPATIAL OFFSETS CALIBRATION Measures and stores 12 calibration factors for the vertical and horizontal registration of each spectral channel
54. atter from the SpeedBeads e Camera Sync Enabled When checked this box begins synchronization of the camera s TDI line rate with the core stream flow speed as measured by the Autofocus Flow Speed system e Remove Background e PMT Settings Displays the PMT settings critical to optimal performance of the Autofocus Flow Speed systems Do not change these values e Fluidics Allows you to control core stream attributes including core diame ter velocity and relative percentage of beads These controls are analogous to those found on the Setup and Fluidics tabs Diameter Allows you to specify the diameter in microns of the hydrody namically focused core stream A larger diameter increases both the object throughput and the positional variability of objects within the core stream The diameter is set to 10 microns for most sample runs Percent Beads Allows you to set the relative contribution of the bead and sample pumps to the core stream Enter the percent of the core stream that consists of beads The proportion of the stream that is not made up of beads is sample During normal operation this is set to 10 beads 90 sample Velocity Allows you to set the core stream velocity Run Runs the fluidics Click to start the sheath bead and sample syringe pumps in proportions that will achieve the desired core stream Diameter Percentage Beads and Velocity This button is analogous to the Play but ton on the Set
55. brightfield light source and orthogonally by a laser s A high numerical aperture NA objective lens collects fluorescence emissions scattered and transmitted light from the cells The collected light in optical space intersects with the spectral decomposition element Light of different spectral bands leaves the decomposition element at different angles such that each band is focused onto 6 different physical locations of one of the two CCD cameras with 256 rows of pixels As a result each cell image is decomposed into six INTRODUCTION separate sub images on each CCD chip based on a range of spectral wavelengths Up to 12 images are collected per object with a two camera system The CCD camera operates in TDI time delay integration mode that electronically tracks moving objects by moving pixel content from row to row down the 256 rows of pixels in synchrony with the velocity of the object cell in flow as measured by the velocity detection system Pixel content is collected off the last row of pixels Imaging in this mode allows for the collection of cell images without streaking and with a high degree of fluorescence sensitivity TDI imaging combined with spectral decomposition allows the simultaneous acquisition of up to 12 spectral images of each cell in flow BASIC ILLUMINATION OPTICS AND IMAGE COLLECTION This section contains information about the ImageStream illumination optics and image collection systems The following d
56. come back into properly focus turn Autofocus tracking on If the cells grad ually become defocused as the focus position moves then Autofocus tracking is not working Run with Autofocus tracking off and call Amnis service Objects are Core stream position is The core tracking and focus tracking objective rotating in the grossly off center within positions should not change significantly from day core stream the flow cell due to air or to day If either value is radically different 20 clog in the fluidics microns objects may rotate due to interactions with the sheath stream An off center core stream is caused by air or clogs in the fluidic system See solution for Air or clog in the sample syringes or flow cell above Everything is Camera synchronization The camera synch value ASSIST tab should not out of Focus setting is incorrect change significantly from day to day If the value is radically different 0 5 re calibrate the camera synchronization using ASSIST or manually set it to a value that has previously worked Advanced Flow Speed tab Excessive core stream See solution for Air or clog in the sample syringes variation due to air or or flow cell clog in the fluidics 108 TROUBLESHOOTING SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Core stream is moving Briefly open the bubble vent Advanced Flow too fast for the camera Speed tab to drop the flow speed to 60 mm sec or lower
57. ction with the Run Acquire mode to capture and image file throughout a focus pan This test particularly helpful to service personnel for the process of characterizing the performance of the instruments Extended Depth of Field capability The utility is not run as part of the routine ASSIST tests Focus Pan Utility Parameters Num Settings Num samples Total Range a i 3 2 a i 0 wo Channel Of Interest r I 1 1 4 5 6 Focus Position um Database Setting Saved Value Result Value 84 INSPIRE M SOFTWARE OVERVIEW PMT Focus UTILITY The PMT Focus Utility provides service personnel with visual and quantitative feedback to characterize the physical alignment and gain settings for each channel of the AutoFocus and Flow Speed system The utility pans the focus stage over a 40 micron range while collecting data at one micron intervals from each channel of the AFFS system The data peaks are determined and fit to 2nd order polynomials to provide a quantitative characterization of the Grating spacing and signal levels for each channel Grating spacing and peak signal levels are reported to the interface The utility is not run as part of the routine ASSIST tests ES PMT Focus Utility Parameters Num Settings Num samples Total Range PMT 1 Pe 0 1 1 1 1 1 1 1 1 1 1 0 1 2 3 4 5 6 7 8 3 10 10 8 6 PMT Energy 4 PMT 2 0 1 2 3 4 5 6 f 8 3 10 10 8 6 4 PMT Energ
58. detection rate Total Sec Displays the per second rate of detection of all objects includ ing cells beads and debris Acquisition status bar Displays the data acquisition progress Below the bar is an indicator light that is green when data acquisition is in progress and a percent complete annotation Elapsed Time A timer that indicates time elapsed since the beginning of data acquisition 50 INSPIRE M SOFTWARE OVERVIEW ILLUMINATION Controls for the brightfield and the fluorescence excitation lasers Brightfield m Excitation Lasers On Off CD On Off Intensity mw 405 50 H Intensity 732 di lt 3 0 Channet 1 gt 488 nm 100 0 4 658 p Set Intensity al lt 120 0 Brightfield Allows control of the brightfield lamp settings On Off Brightfield power button Channel Drop down menu that allows you to direct brightfield light to the specified camera channel Once you select a channel the brightfield background intensity is set to the desired level Intensity The current value for the brightfield background intensity Set Intensity Delivers the desired amount of brightfield light to the cam era The default amount achieves a background mean pixel intensity of 200 counts on the camera Excitation Lasers Allows control of the indicated fluorescence excitation lasers On Off Turns lasers on or off Intensity mW Allows variable control over
59. e run Flush Lock Load with a solution of 10 bleach and then PBS to ensure that residual dye does not stain the subsequent samples 20 Name the new file in the Data File window 28 OPERATING THE IMAGESTREAM USING INSPIRE 21 22 23 24 25 26 27 28 Center the core stream images if necessary by laterally moving the objective under Core Tracking Ensure that the Flow Speed CV is consistently less than 0 2 by monitoring the Flow Speed CV graph on the Setup tab Click Cell Detection and double check the cell classifiers Click Run Acquire Repeat for each sample When finished running all of the experiment samples click Flush Lock Load and load 60 ul of 10 bleach then PBS solution to clean out any contaminating dye if necessary Press Flush Lock Load and run the first compensation control For compensa tion controls the following settings are critical turn brightfield and scatter 785 nm laser OFF check every channel to be collected in the cell classifier window and keep all laser powers the same as for the experimental sam ples Using Flush Lock Load continue collecting the compensation controls OPTIONAL SETTINGS SETTING THE IMAGE DISPLAY PROPERTIES 1 2 Click on one of the channel configuration boxes Select the color gain and log or linear transformation for image display of each channel The color is also applied to data points in the Acquisition Plots Auto set Thresholds Backgro
60. e ensquared energy ratio in each image The ensquared energy for each image at each focus location is shown in a plot The mean ensquared energy for each focus position is noted as a dark blue data point for each focus position The ensquared energy for the top 2 of all imagery is computed and indicated as a dark blue data point on the plot This result is tested against predetermined limits and reported on the Collection Image Quality test tab and in the popup window This value is stored in the ASSIST database A highly magnified composite image of the top 2 of all images is also generated and displayed on the popup window Each small square of light is an individual pixel approximately 0 5 microns on a side in object space This image generally shows a small amount of flair on the right hand side This is due to light scatter from the far side of the SpeedBead which is approximately 1um in diameter INSPIRE SOFTWARE OVERVIEW 77 i Image Quality 40x Test Parameters Num Settings Channel Of Interest Num samples Total Range EE Image Quality 0 82741 5 0 75 06 05 0 4 Ensquared Energy 03 02 0 14373 a y J Focus Position um Database Setting Saved Value Result Value ASSIST CollectionImageQualityFocusTest_LimitMin 0 55 ASSIST Collection mageQualityFocusT est_LimitMax 1 00 ASSIST CollectionlmageQualityFocusT est_PercentUsed 0 02 ASSIST CollectionlmageQualityFocusT es
61. eam system During sterilization the system e Turns off all illumination sources e Empties all syringes and fills the sheath syringe with de ionized water e Rinses the entire fluidic system with water sterilizer cleanser debubbler and water in that order The sterilizer is held in the sys tem for 10 minutes to ensure decontamination before the process refills the system with the next reagent e Empties all three syringe pumps Purge Bubbles Removes air bubbles from the flow cell by filling the flow cell with air then filling the sheath line and pump with debubbler and rins INSPIRE SOFTWARE OVERVIEW 41 ing the flow cell The sheath syringe is then refilled with sheath and the bubble trap lines and flow cell are filled with sheath Dual Prime Rapidly delivers both the cell sample and SpeedBeads to the flow cells in the proportion set in the Core Size and Speed area of the Setup tab Service Scripts Condition Valves Exorcise Syringe For field service per sonnel only e Sample menu Run fluidic scripts specific to the sample pump Sample Flush Lock And Load Sample Load Sample Prime Sample Return Sample Flush Lock And Load Sample Backflushes the sample and bead lines and rinses the sample syringe with sheath fluid three times introduces a 50 ul air bubble loads and primes the sample to deliver the proportion of beads and sample material that is set in the Core Size and Speed area of the
62. ec This speed corresponds to the highest resolution setting with a pixel size of 0 5 um In order to collect images at higher sensitivy the rows on the camera can be binned 2X binning mode combines 2 rows on the camera into one digit The same amount of total intensity is collected Image resolution and sensitivity are inversely related to one another DAILY SHUTDOWN PROCEDURE This procedure sterilizes the system and leaves it with pumps empty and water in the fluidic lines The instrument is left on with INSPIRE running 1 2 3 4 Fill the Rinse Cleanser Sterilizer and Debubbler bottles if necessary Empty the Waste bottle Remove any tubes from the uptake ports Click Sterilize Init Fluidics DE Script Note This procedure automatically turns off all illumination sources and rinses the entire fluidic system with water sterilizer cleanser de bubbler and then water again The sterilizer is held in the system for ten minutes to ensure de contamina tion It takes about 45 minutes of unattended walk away operation to complete FULL SHUTDOWN PROCEDURE If the ImageStream will not be used for more than two weeks follow the daily shutdown procedure above and in addition clean and store the bead line in water OU A N e Follow the daily shutdown procedure above Remove the bead vial from the instrument Replace with a vial of DI water Run Initialize fluidics script Choose Exit and shutdown instrument f
63. ed e Brightfield in channel 4 is set to 800 counts e 785 nm laser is on and power is set to 2 3 mW e Core stream velocity is set to 60 66 mm sec e Core diameter is set to 6 8 microns e Percent beads is set to 100 20 OPERATING THE IMAGESTREAM UsiNnG INSPIRE 7 Click Initialize This script fills the system with sheath and flushes out all of the old sheath or rinse that was in the system The sample line is prepared by loading 50 ul of air into the uptake line Beads are loaded into the bead pump from the 15 ml conical tube At the end of this script SpeedBeads should be running and the flow speed amplitude graph which displays the SpeedBead signal can be observed Flow Speed Amplitude 4 3 2 1 0 1 221 0 20 40 60 86 Core Velocity mm sec 8 Click Run Setup to start the camera and select All from the View dropdown box to start imaging the beads ES INSPIRE for the imageStream Pie Iretrumert Sample Leeds Help Run amp cqure ch 9 Center the core stream images if necessary by laterally moving the objective under Core Tracking Core Tracking is adjusted by pressing right or left arrows to center images set increment to 5u Observe the Flow Speed Amplitude plot on the Setup tab If a core stream has formed a dominant overlapping red and black peak is displayed in the plot if not see Troubleshooting on page 103 OPERATING THE IMAGESTREAM USING INSPIRE 21 10 In a few seconds the Flow S
64. est 10 28 2009 8 52 39 PM 488nm Laser Power Test 10 28 2009 8 52 52 PM 658nm Laser Power Test 785nm Laser Power Test 10 28 2009 8 53 02 PM 10 28 2009 8 53 14 PM Brightfield Intensity Selection Test Brightfield Uniformity Test 10 28 2009 8 53 44 PM 10 28 2009 8 54 12 PM Flow Core Axial Stability Test 10 28 2009 8 54 36 PM Flow Core Lateral Stability Test 10 28 2009 8 54 53 PM Flow Core Position Test 10 28 2009 8 55 15 PM Focus Offset 40x Test 10 28 2009 8 57 34 PM Image Quality 40 Test 10 28 2009 8 57 58 PM Utilities Utilities Last Run Time Brightfield Calibration Utility 10 28 2009 9 02 02 AM EDF Excitation Utility 10 15 2009 4 28 33 PM Focus Pan Utility PMT Focus Utility 10 28 2009 9 26 16 AM 10 28 2009 10 54 42 PM SCurve Peaks Utility 10 28 2009 5 48 54 PM History Start All Calibrations and Test 58 INSPIRE SOFTWARE OVERVIEW Stop All ASSIST CALIBRATIONS The calibrations in the current suite are described in detail below CAMERA SYNCHRONIZATION CALIBRATION Measures and stores a magnification calibration camera synch factor relating the Flow Speed Detection frequency and the camera clock rate This factor is used to maintain synchronization between the moving imagery projected onto the camera surface and the electronic charge resulting from that imagery Proper synchroniz
65. f all the objects that are detected in the Cell Bead and Debris classifications 100 INSPIRE SOFTWARE OVERVIEW Cells Second Beads Second Debris Second Displays the cell bead or debris detection rate Total Second Displays the per second rate of detection of all objects including cells beads and debris Setup ASSIST Advanced Setup Advanced Fluidics Autofocus Flow Speed Camera Illumination Display Acquisition Object Detection Cells to Acquire 5000 M Auto Detect Beads Bead Classifier Squelch a Auto BF Gain 4 00 v Correction Data File Folder Z Temp nbauersfeld Browse File Name Filename Sequence Number h D Save Bead Image Data D Save Debris Image Data Path Acquisition In Process Cell Count EL Bead Count a Debris Count i Total Count E Cells Second 560 Beads Second g Debris Second E Total Second 560 101 INSPIRE SOFTWARE OVERVIEW AUTOSAMPLER TAB The Autosampler tab allows manual manipulation of the autosampler These controls are to be used under the guidance of Amnis service personnel only 102 INSPIRE SOFTWARE OVERVIEW CHAPTER 5 Troubleshooting This chapter is designed to help you troubleshoot the operation of the ImageStream If additional assistance is required contact the Amnis service department The troubleshooting guide is broken up into the following topics e No flow speed signal e Flow speed sig
66. han 10 and must meet transient over voltage category II The instrument is evaluated to Pollution Degree 2 Noise level The noise level of the ImageStream is less than 70 dB A Weight 160 kg Ventilation Provide at least 3 inches of clearance behind the instrument to maintain proper ventilation Disconnection To disconnect the instrument from the power supply remove the plug from the socket outlet which must be located in the vicinity of the machine and in view of the operator Do not position the instrument so that disconnecting the power cord is difficult To immediately turn the machine off should the need arise remove the plug from the socket outlet INFORMATION AND SAFETY 1 2 Transportation The ImageStream relies on many delicate alignments for proper operation The machine may be moved only by an Amnis representative Cleaning Clean spills on the instrument with a mild detergent Using gloves clean the sample portal and sample elevator with a 10 bleach solution Dispose of waste using proper precautions and in accordance with local regulations Preventative maintenance The ImageStream contains no serviceable parts Only Amnis trained technicians are allowed to align the laser beams or otherwise repair or maintain the instrument The instrument fluidic system is automatically sterilized after each day s use This reduces the occurrence of clogging Tubing and valves are replaced by Amnis service person
67. he ability to invoke a single test by clicking a button The four tests in the current suite are described in detail below EXCITATION LASER POWER TESTS The power of each excitation laser present in the system is measured and tested against limits by quantifying the amount of light scattered from SpeedBeads The instrument is configured specifically to test each laser by adjusting classifiers setting stage selections and inserting the proper neutral density filters into the collection path The test compares the mean signal strength acquired from each laser and compares it to radiometric ally calibrated signal strengths collected during the manufacturing process The intensity of each laser is stored in the database The results and limits of the test are shown below the list when the test is selected 68 INSPIRE SOFTWARE OVERVIEW BF INTENSITY SELECTION TEST Verifies the BF intensity calibration for each BF mode The image intensity must reach 200 within 20 iterations If this test fails the user should run the BF Intensity Selection Calibration individually and then re run the test Brightfield Intensity Selection Test Parameters Num Settings Channel Of Interest Num samples Total Range Chann a 2 w 8 5 5 F j 3 Channel REWER SUMBANME AMARA e Idle Database Setting Saved Value Result Value ASSIST BFTestQualityMin 0 00 ASSIST BFTestQualityMax 20 00 ASSIST BFTestQualityCh1
68. he responsibility of the end user Follow all local state and federal biohazard handling regulations for disposal of the contents of the waste reservoir Prevent waste reservoir overflow by emptying the container when the waste indicator indicates that it is full Run the instruments sterilize routine after each day s use Note that this procedure has not been proven to result in microbial sterility The biosafety Level for the instrument is BSL1 SECURITE BIOLOGIQUE Biorisques L image Stream est valu un niveau de s curit biologique L1 Ne pas acqu rir ou vider des chantillons contenant des agents infectieux sans les avoir inactiv s Il est recommand que les chantillons soient fix s dans du paraformald hyde 2 pendant au moins 10 minutes avant d acqu rir des chantillons avec l ImageStream L utilisation le confinement et l limination des mat riels biologiques dangereux sont tenus d tre en conformit avec les normes de s curit relatives au laboratoire et de la directive 93 95 E et restent sous la responsabilit de l utilisateur Respectez la r glementation en vigueur pour le traitement et l limination des d chets dans des r servoirs pr vus cet effet Pr venir l accumulation des d chets en vidant le r servoir lorsque l indicateur indique qu il est plein St riliser les instruments de routine apr s chaque journ e d utilisation Notez que cette proc dure ne garantit pas la st rilit vis
69. history Fluid Levels Reports information from the ImageStream fluid sensors When one or more of the fluid levels are low or if the waste tank is full the indicator light will turn red and a pop up window appears that indicates which fluid tank needs to be filled or emptied Note Wait for a script to finish before refilling the sheath tank to avoid introducing air into the system Syringe Volumes Indicates the volume in the Sheath Sample and Bead syringe pumps Mouse over the syringe name to see the volume in microliters Flow Speed Amplitude Plot that displays the core stream velocity in mm sec corresponds to the Actual velocity vs signal strength as measured by the PMTs represented in red and black The frequency of signals received through the gratings by the PMTs from an object is related to the core stream velocity in mm sec The focal position of the core stream is calculated from the balance of signal amplitudes of the two PMTs If the core stream drifts in the z axis the amplitude measured by one PMT will increase while the amplitude measured by the other will decrease INSPIRE SOFTWARE OVERVIEW 55 Flow Speed CV Plot that displays the variation in the measured core stream velocity as a percentage of the mean sample speed coefficient of variation CV stream over time fluidically stable core stream will have flow speed CV s consistently below 0 2 Higher values can result in image streaking Note Doub
70. iagram is a schematic of these systems ImageStream System Optical Layout Main subsystems shown in color are the Brightfeld Illumination Image Collection camera one Image Collection camera two AutoFocus Flow Speed System Excitation Lasers INTRODUCTION 11 BRIGHTFIELD ILLUMINATION Brightfield illumination of the sample is achieved using a spectrally filtered LED that transilluminates objects in the flow cell The LED emission wavelength and spectral filter arrangement is designed to optimize the spectrum of light appropriate for the camera channel to be used for brightfield image collection The LED illuminator is designed with 6 separate LEDs that are spectrally multiplexed to allow multiple channels of brightfield to be collected simultaneously LASER EXCITATION OPTICS Fluorescence excitation is achieved with a variable power 10 to 100 mW solid state 488 nm laser Optional fluorescence excitation lasers include a higher power 500 mW 488 nm laser a 125 mW 405nm laser a 200 mW 561 nm laser a 300 mW 592 nm laser and a 120 mW 658 nm laser A 70 mW 785 nm laser provides a scatter signal and is collected in channel 6 Brightfield laser scatter and fluorescent images are collected on six channel charge coupled device CCD camera s run in time delay integration TDI mode An optional second camera enables 12 channel image collection Light is collected from the cells flowing in the cuvette with a microscope objective and re
71. ibility Part 4 Testing and Measurement Techniques Section 4 Electrical Fast Transient Burst Immunity Test EN 6100044 2004 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 5 Surge Immunity Test EN 61000 4 5 2005 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 6 Immunity to Conducted Disturbances Induced by Radio Frequency Fields EN 610004 6 2008 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 8 Power Frequency Magnetic Field Immunity Test EN 61000 4 8 2001 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 1 1 Voltage Dips Short Interruptions and Voltage Variations Immunity Test EN 610004 11 2004 edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 1 2001 2 edition AUTHORIZED REPRESENTATVE Izasa S A Aragon 90 08015 Barcelona Spain VAT ESA281 14742 Contact Jose Maria Alonso 34 916 630 550 TEST FACILITY F Squared Laboratories Northwest EMC 26501 Ridge Road 22975 NW Evergreen Parkway Ste 400 Damascus MD 20872 USA Hillsboro OR 97124 USA The Cellular Analyzer Model ImageStream X is in effective conformance to the Directives and Standards referenced above Authorized wk amp Date 1 September 2009 Name Richard Esposito Title Quality Assu
72. ifted from pixel to pixel down the detector parallel to the axis of flow By synchronizing the photocharge shift rate with the velocity of the flowing cell the effect is similar to physically panning a camera With TDI image streaking is avoided despite signal integration times that are orders of magnitude longer than those of conventional flow cytometry Each pixel is digitized with 12 bits of intensity resolution providing a minimum dynamic range of three decades per pixel INTRODUCTION 13 AUTOFOCUS AND FLOW SPEED SYNCHRONIZATION The ImageStream continuously and automatically adjusts for small variations in the flow rate and the focal position of the cells in the flow stream during sample runs The system uses a reagent SpeedBeads and an auxiliary 830 nm diode laser and PMTs SpeedBeads are 1 micron beads that are continuously run through the flow cell during the operation of the ImageStream Light scattered off of the beads from the 830 nm laser is collected through the objective lens and directed into the Autofocus Flow Speed optical system This system optically conditions this light and focuses it through a series of gratings and into photomultiplier detectors The signal from the photomultipliers is further processed to generate both a flow speed control signal and a focus error signal The flow speed signal is used in a control loop to constantly update the camera line rate to maintain image synchrony Likewise the focus error
73. in 3 Linear Threshold Log Gain 4 Linear Threshold Log Gain 5 Linear Threshold C Log Gain n CE eaS r Linear Threshold 0 C Log Gain r Linear Threshold 0 C Log Gain n Linear Threshold 0 C Log Gain p Linear Threshold 0 C Log Gain ces Linear Threshold 0 C Log Gain m E Linear Threshold Linear Threshold 0 Color Log Gain Log Gain 6 El Thresholds Auto set Thresholds Default Thresholds Saturated Color Reset All Channels Color INSPIRE SOFTWARE OVERVIEW 45 IMAGING AREA AND TOOLS The imaging area of the INSPIRE screen occupies the majority of the left side of the screen It displays the channel images of the beads and or sample Immediately to the left of the imaging area are tools that allow you to perform detailed image analysis The imaging area and tools include the following View dropdown box Determines whether cells beads debris or all three are displayed in the imaging area besc Fm e Magnifying glass icons Zoom in or out on the imagery The scroll bar at the bottom of the image window can be used to see out of view channel images 8 e The arrow line and box open the Image Display Tools window for detailed pixel intensity analysis of the imagery CL 4 Ptr Line Rgn Buttons that allow interrogation of pixel information of a single point Pt
74. ion of Conformity Explanation of SyMbols der Rene En ta SN LT Electrical Safety sant ssh gph deeb hy beet eee de 2 S cunte Electronique s m rose nement Mere Baa Ares Laser Safety castes sop sng desu dude RPM en man ata ne ee R S S CULIL LASER Aik fe eet We A oe We Se E Sido Lt Biological Safety ss bee atecaes de EE E Has re eds ear dacs S curit Biologique sesen A pene iai Lt hee ede Lies Spare Parts Lists peurs ans i ie rat ies eae Ae ey ae ee nie SaaS INTRODUCTION Introduction to the ImageStream Technology Overview ss sg Lane rar sr et oa dee ete Slew Basic Illumination Optics and Image Collection SpeedBeads and Cell Sampless in siaa e Ree dee es a EE ENE N OPERATING THE IMAGESTREAM Us nG INSPIRE NETT MOSA A a ES S ee ie et a EAO RAAE DNE A AR a INSPIRE User Interfaces ors seu dune aiaa en et dot Daily Calibration and Testing Datar A quisition sesir seize hee bugged Genes weeks OA waa ok eae Daily Shutdown Procedure Optional upgrades INSPIRE SOFTWARE OVERVIEW INSPIRE User Interface ta n paare ei erni ii eae ete eee ede Men Baris cad tans an Meret dans le dees wees AGHON Buttons 4 032 4464 desde ant sie ged cettewdete aga Configure Channel Display Settings Imaging Arearand Tools ruei anne Me ahd tes Sate E A R
75. is off Note If the ImageStream power is on and INSPIRE is already launched go directly to step 4 Press the green power button inside the front door of the ImageStream to turn on the instrument and start the computer Log on with the user name Amnis and password IS100 Launch the INSPIRE software and by double clicking the INSPIRE icon on the desktop PREPARING TO RUN CALIBRATION 4 Fill the rinse bottle with deionized water and the sheath bottle with PBS Ensure the SpeedBead reagent is loaded on the bead port and is well mixed The beads are automatically mixed while the instrument is in use If the instrument has been idle for a long period remove the bead vial and vortex Refer to the following compat ibility chart to choose the appropriate Sheath fluid Pull and lift the black knob to lift the waste line out of the container Add 160 ml of bleach to the Waste bottle The final volume of waste when full will be 1600 ml and therefore the final bleach concentration for a full waste tank will be 10 bleach It is recommended that the waste be emptied every day and fresh bleach added before Intitializing fluids From the File menu select Load Default Template This turns on the 785 laser and brightfield illumination sources and configures the instrument for ASSIST Verify that the following critical settings all of which you can set on the main screen and the Setup tab are in place e Imaging is off click Stop if enabl
76. ith well A1 positioned at the upper left corner 15 Click Start to begin 16 The Status column will be updated for each well as it is run For each sample the instrument performs the following in sequence 1 Flush Lock and Load 2 Vali dation flow speed CV focus brightfield intensity object rate 3 Data Acquisition 4 Result success or error 17 During a run e You may stop the plate at any time by clicking the Stop button This does not initiate sterilize even if the Sterilize after running plate box is checked 37 OPERATING THE IMAGESTREAM USING INSPIRE e Should the sheath tank or beads reservoir become empty or the waste tank full during a run an alert will be sent to the email entered in the well plate defini tion Acquisition will pause until the user intervenes e Ifan error occurs on a well the sample is returned an alert is sent to the email address entered in the well plate definition and the autosampler moves on to the next well Ifthe same error occurs on three consecutive wells the autosampler aborts the plate and sterilizes the instrument if the Sterilize after running plate box is checked 18 A report will be saved to the folder designated in the Output File Path of the plate definition at the end of the run either when it was stopped manually or completed the entire plate Well Parameters Standard Parameters Fluidics Parameters Date O Percent Beads Hwe
77. layed to the spectral decomposition element filter stack which consists of a set of wavelength selective filters in an angular array The filter stack directs different spectral bands to laterally distinct channels on the detector With this technique an image is optically decomposed into a set of six sub images per camera each corresponding to a different color component and spatially isolated from the remaining sub images A high NA 40X objective lens is standard The optional MultiMag upgrade provides both 20X and 60X imaging objective lenses The spectral bandwidth of each camera channel is given in the tables below TABLE 1 1 CAMERA 6 CHANNELS CHANNEL WAVELENGTH NM 1 430 505 2 505 560 3 560 595 4 595 660 5 660 745 6 745 800 12 INTRODUCTION TABLE 2 2 CAMERAS 12 CHANNELS CHANNEL WAVELENGTH NM 1 430 480 2 480 560 3 560 595 4 595 660 5 660 745 6 745 800 7 430 505 8 505 570 9 570 595 10 695 660 11 660 745 12 745 800 IMAGE COLLECTION The CCD is operated using a technique called time delay integration TDI a specialized detector readout mode that preserves sensitivity and image quality even with fast relative movement between the detector and the objects being imaged As with any CCD image photons are converted to photocharges in an array of pixels During TDI operation the photocharges are continuously sh
78. le click on a graph to launch the Chart Properties window which allows you to customize the graph display 56 INSPIRE M SOFTWARE OVERVIEW ASSIST TAB ASSIST Automated Suite of Systemwide ImageStream Tests is a suite of calibrations and tests for critical subsystems operating within the ImageStream ASSIST performs specific calibrations and tests measuring evaluating and storing thousands of values to ensure all subsystems are operating within normal limits ASSIST permanently logs results for all tests and flags any parameters that are beyond specified limits It is run daily using SpeedBeads to ensure optimal performance of the ImageStream A calibration is a sequence of operations designed to measure and set internal parameters that are used to operate a subsystem Calibrations are used to optimize performance of a subsystem or place it in predefined state After a calibration is performed it is tested to determine whether the calibration values are within a prescribed range A test is a sequence of operations designed to measure the performance of a specific subsystem The calibration and test values and acceptable ranges are listed on the ASSIST display tab A failed calibration or test is flagged with a red box The history of any calibration or test can be viewed by clicking on the box to the right of the specific item Utilities are calibrations used by service technicians Run the Brightfield Calibration Utility if the Brightfield
79. luid and flushes the instrument with the new sheath fluid The sample and the SpeedBeads are also flushed out of their respective pumps The sample uptake line is loaded with 50 ul of air in preparation for a sample load SpeedBeads are loaded into the bead syringe pump and primed to the flow cell establishing a core Sterilize Decontaminates and cleans the ImageStream system During sterilization the system Turns off all illumination sources e Empties all syringes and fills the sheath syringe with de ionized water e Rinses the entire fluidic system with water sterilizer cleanser debubbler and water in that order The sterilizer is held in the sys tem for ten minutes to ensure decontamination before the process refills the system with the next reagent e Empties all three syringe pumps Abort Script Stops a fluidic script that is running The button is enabled only when a script is running 44 INSPIRE SOFTWARE OVERVIEW CONFIGURE CHANNEL DISPLAY SETTINGS The Channel names and display intensities can be adjusted by opening the Configure Channel dialog Click on any channel number to open the Configure Channel dialog Click on a color swatch to change the color display Adjust the gain of the display by entering a number in the Gain box Note these settings are for display only and do not effect the intensity values Configure Channel Linear Threshold Log Gain Linear Threshold Log Ga
80. n Sample Dual Prime Prime Beads Initialize Fluidics Sterilize Left Control camera setup and acquisition Middle Fluidic scripts for running samples Right Fluidic scripts for startup and shutdown procedures e Imaging Area and Tools _Observe imagery and manipulate image display e Acquisition Plots Real time graphs of calculated image parameters such as size and pixel intensities used to set classifiers to identify objects for inclusion in the data file Cells are plotted as green dots The right side of the window contains the following three tabs Setup The user interface for routine operation of the ImageStream e ASSIST The user interface for running instrument calibrations and tests e Advanced Setup Additional tabs that allow manual control for service and troubleshooting over the fluidics autofocus flow speed camera and illumina tion subsystems and control of imaging display and acquisition MENU BAR The menu bar is located in the upper left portion of the INSPIRE screen It consists of these four menus e File menu Load and save instrument setup templates A template contains instrument settings that can be predefined and loaded to simplify the instru ment setup process Fle Open Template Ctr 0 Save Temelate Load Default Template Exit and Shutdown Instrument Exit Open Template Browse for and open saved templates 40 INSPIRE SOFTWARE OVERVIEW Save Template Sa
81. nal disappears No images e No cell images e No bead images e Ratio of bead to cell images is higher than expected e Cell Classifiers are not working e Imaging is intermittent or appears frozen and object acquisition rates are erratic e INSPIRE appears to freeze e Images appear streaked e Cells are not centered in the channel e Core stream is not in the normal core or focus tracking range e Objects are rotating in the core stream e Everything is out of focus e Flow speed is not stable e Flow speed changes rhythmically e Fluidics respond sluggishly e Core stream velocity is too high or low after a prime e Actual velocity reading appears frozen e Event rate slows over time Flow rate slows or stops over time e Fluorescence imagery appears too dim e Everything is too bright or images are red or flat in appearance e One channel saturates while the others do not Cross contamination from previous samples Scatter control value set by ASSIST is abnormally low Scatter is too dim or bright Scatter intensity changes over time e Large variation in brightfield intensity levels Brightfield intensity level sets incorrectly TROUBLESHOOTING 103 e Blue non parsed imagery in all channels e Erroneous waste sensor low indicator Instrument will not pass ASSIST SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS No flow speed signal Objective stage position is incorrect Move the objec
82. nd if it fails call Amnis service 405 488 658 785 Laser Power test failure Run the test a second time and if it fails call Amnis service Brightfield Intensity Selection test failure Run the test a second time and if it fails call Amnis service TROUBLESHOOTING 113 SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Brightfield Uniformity test failure Run the test a second time and if it fails call Amnis service Flow Core Axial Stabil ity test failure Verify that the SpeedBeads are running well and that there is no air trapped in the system Run the Purge Bubbles or Sterilize System scripts then repeat ASSIST Call Amnis service if it continues to fail Flow Core Lateral Stabil ity test failure Verify that the SpeedBeads are running well and that there is no air trapped in the system Run the Purge Bubbles or Sterilize System scripts then repeat ASSIST Call Amnis service if it continues to fail Flow Core Position test failure Verify that the SpeedBeads are running well and that there is no air trapped in the system Run the Purge Bubbles or Sterilize System scripts then repeat ASSIST Call Amnis service if it continues to fail Focus Offset 40X test failure Verify that the SpeedBeads are running well and that there is no air trapped in the system Run the Purge Bubbles or Sterilize System scripts then repeat ASSIST Call Amnis service if it
83. nel as part of a routine preventive maintenance schedule Access to moving parts The movement of mechanical parts within the instrument can cause injury to fingers and hands Access to moving parts under the hood of the ImageStream is intended only for Amnis service personnel Protection impairment Using controls or making adjustments other than those specified in this manual can result in hazardous exposure to laser radiation in exposure to biohazards or in injury from the mechanical or electrical components FCC compliance This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to part 15 of the FCC rules These limits were designed to provide reasonable protection against harmful interference when the equipment is used in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual can cause harmful interference to radio communications The operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at the user s own expense INFORMATION AND SAFETY DECLARATION OF CONFORMITY IILIXILIIIIIIILILIXIIIIIIILILINIIILIIIIILIANIIIIIILIXIKIIIIILILILINIIIIILILIXERIIILILILILIXIIIIILIXINIIIIIIT DECLARATION OF CONFORMITY IN ACCORDANCE TO ISO IEC GUIDE 22 FORA Cellular Analyzer MANUF
84. ng EDF on page 31 for details Set Cell Classification criteria SETTING CELL CLASSIFIERS You can use the Cell Classifier window to set cell classifiers and to select channels for collection Select channels collected by checking unchecking the box under the channel Note It is critical to collect all channels for compensation control files To identify objects for inclusion in or exclusion from the acquiring data file set upper and lower limits for the following features in any of the six channel images Area The number of pixels in an image reported in square microns e Max Gradient Intensity The value of the largest slope spanning three pixels in an image This feature measures image contrast or focus quality Intensity The integrated intensity of the entire object image the sum of all pixel intensities in an image background subtracted e Mean Pixel The average pixel intensity in an image background subtracted e Raw Max Pixel The intensity value of the brightest pixel in an image e Raw Min Pixel The intensity value of the dimmest pixel in an image Saturation Count The number of pixels in an image that have an intensity value of 4096 e Saturation Percent The percentage of pixels in an image that have an inten sity value of 4096 Objects that fall above an upper limit or below a lower limit are considered debris by the instrument and will be excluded from the file Common examples include
85. ng enabled Activates the feature that automatically adjusts the z axis position of the objective to find and maintain optimal focus 52 INSPIRE SOFTWARE OVERVIEW FLUIDICS Information about and control of the fluidics system is provided in several areas of the Setup tab Fluidics Diameter microns Percent Beads Core Velocity mm sec Desired Actual 66 2565 e Fluidics and Core Velocity controls Diameter Allows you to specify the diameter in microns of the hydrody namically focused core stream A larger diameter increases both the object throughput and the positional variability of objects within the core stream The diameter is set to 10 microns for most sample runs Percent Beads Allows you to set the relative contribution of the bead and sample pumps to the core stream Enter the percent of the core stream that consists of beads The proportion of the stream that is not made up of beads is sample During normal operation this is set to 10 beads 90 sample Core Velocity Allows you to control the core stream velocity Desired Allows you to set the desired speed of the core stream This is typically set to 66 mm sec to achieve maximum object throughput without outpacing the ability of the camera to track the objects e Actual Displays the current velocity of the core stream e Play button Run Runs the fluidics Click to start the sheath bead and sample syringe pumps in proportions
86. ng the 20X objective is 1 square micron The 60X objective provides a higher magnification for small objects The pixel size using the 60X objective is 0 33 microns Objective Field of view Pixel size Depth of field NA 40X 60 um 0 5 um 4um 0 75 20X 120 um 1 um 8 um 0 5 60X 40 um 0 33 2 5 um 0 9 The optional objective can be chosen by selecting the button under Magnification When using the 60X obective the core velocity will be reduced to 40 mm sec instead of the normal 60 mm sec used during 40X or 20X acquisition 32 OPERATING THE IMAGESTREAM USING INSPIRE USING THE AUTOSAMPLER To enable high throughput experiments and unattended operation the autosampler option includes upgraded fluidics software and an imbedded nest for loading of samples in a 96 well plate format Prior to running the plate a plate definition is created that assigns instrument settings to the wells names to the output files and parameters to include in a well plate report that is generated once the plate has completed While the plate is running the user is notified of any errors encountered via email The instrument can also sterilize at the completion of the plate WORKFLOW Create Instrument Setting Template s ist to be used for your plate To do this run an experimental sample manually with all of the fluorescence dyes to be used in the experiment see INSPIRE Setup Quick Start Guide Save each relevant template Create a Well Plate
87. nted in the red and black Time Series graphs The flow speed is calculated from the fast Fourier Transform of the time based PMT signals The frequency of signals received through the gratings by the PMTs from an object is related to the core stream velocity in mm sec The focal position of the core stream is calculated from the balance of signal amplitudes of the two PMTs If the core stream drifts in the z axis the amplitude measured by one PMT increases while the amplitude measured by the other decreases Bead Concentration vs Time The concentration of beads measured by both PMT channels red and black of the Autofocus Flow Speed system Used by Field Service for diagnostic purposes only 90 INSPIRE M SOFTWARE OVERVIEW Setup ASSIST Advanced Setup Advanced Fluidics Autofocus Flow Speed Camera Illumination Display Acquisition Flow Speed Vv Tracking Enabled Automatic Core Tracking On Off Focus Tracking Absolute gt 284 Current Relative 4 2 Default w Auto Focus QJ Tracking Enabled Min Time Between Updates in seconds 120 Core Tolerance in fractions of channel width 0 25 274 1 Set Default 275 4 Go Default 000 gt AutoFocus Error Notification none PMT Settings Focus Offsets A loss FiterLenath 10 K foo B fois Focus Hyst Comp At Reverse Limit Tracking aaa Hyst Comp ore Tracking 0000 4 Absolute gt 2000 Curren
88. ntsChan2 0 06 1 00 0 ASSIST BFCrossT alkCoefficientsChan3 0 07 0 04 1 INSPIRE SOFTWARE OVERVIEW 63 HORIZONTAL LASER CALIBRATIONS The alignment of each laser in the ImageStream is automatically controlled to ensure optimal performance via the Horizontal Laser Calibration The calibration routine sweeps the horizontal position of the laser across the flow stream At each of 15 predefined intervals during the sweep 1000 SpeedBead images are collected and analyzed to determine the intensity of each bead The median intensity for each position is then plotted and fit to a fourth order polynomial The peak height of the polynomial is then determined This position is the point where the peak intensity of the Gaussian laser beam intersects the center of the flow core This position provides both the highest intensity for illuminating the core stream and the point with the lowest coefficient of variation This position is stored for each laser and used as the default position during subsequent assays 405 Horizontal Laser Calibration Parameters Num Settings Channel Of Interest Num samples FF Total Range 62315 50000 40000 30000 Total Intensity 20000 10000 5015 7 1 1 1 1 60 40 20 0 20 40 60 Horizontal Laser Position um Idle Database Setting Saved Value Result Value ASSIST ExcHarizontalL aserPosition_405 4 95 AB ASSIST ExcPowerT est_LaserPower_405 3 00 64 INSPIRE S
89. of the ImageStream Many assays that are run on the ImageStream quantify the spatial relationships between molecules located within cells of interest To accurately perform these measurements and to accurately perform spectral compensation of image data the ImageStream must maintain sub pixel spatial registry between channels Spatial Offsets Calibration Parameters Num Settings Channel Of Interest Num samples ia Total Range Changing Instrument Settings Database Setting Saved Value Result Value ASSIST XOffsets_Gen2 0 04 0 05 0 ASSIST Offsets_Gen2 0 00 0 04 ASSIST SpatialO fisetsMin 0 95 ASSIST SpatialOffsetsMax 0 95 8SSIST SpatialOffsets2ndRefMin 5 00 ASSIST SpatialOffsets2ndRefMax 3 00 Chi amp Offset The SpatialOffsets calibration commands the brightfield system to illuminate all 6 channels simultaneously and collects imagery from 1000 SpeedBead objects in each of the six channels 6000 images total It then performs a two axis autocorrelation between the imagery from channels 1 5 with the imagery from channel 6 Autocorrelation is an accurate algorithmic technique that identifies the point at which two images exhibit the highest degree of overlap The autocorrelation results in a vertical and horizontal coordinate for each image correlation These values are then processed to determine the mean coordinates to bring each channel into spatial registry with channel 6 and therefore with each other
90. onvolved to create an image of the entire cell projected simultaneously in focus e During acquisition and before deconvolution images will appear blurred into characteristic L shaped patterns and raw max pixel values will be lower with EDF than with standard mode collection e Compensation controls for EDF data can be collected with or without the EDF element in place e When analyzing data in IDEAS after the deconvolution process there will be more light per pixel than in non deconvolved imagery Therefore raw max pixel values may exceed 1023 for the IS100 instrument or 4095 for the ISX As long as the images did not saturate the camera during acquisition these pixel values are valid e Object Morphology and System Masks will be smaller in EDF mode 31 OPERATING THE IMAGESTREAM USING INSPIRE e Focus gating is not required However if there are blurred events due to streak ing these can be removed from the analysis using a focus gate EDF images exhibit increased texture due to higher resolution Brightfield imagery 1s not as crisp in EDF mode as in standard mode e An in depth discussion of EDF can be found in the following reference Cytometry Part A 2007 71A 215 231 USING MULTIMAG The MultiMag option includes 2 additional objective lenses The 20X lense is useful for very large objects that do not fit into the field of view of the 40X objective such as cardiomyocyes or epithelial cells The pixel size usi
91. osition Default Displays the default position of the objective stage in the x axis Click Go to Default to move the objective to this position At Reverse Limit Turns green when the objective stage reaches the limit of its mechanical range of motion in the x axis INSPIRE SOFTWARE OVERVIEW 89 e Graphs All six graphs on this tab report PMT based measurements of the Autofocus Flow Speed system that relate to Focus Tracking Note Double click on a graph to view the Chart Properties window which allows you to adjust the display properties Autofocus Stage Position Displays the z axis objective stage movement in microns um relative to the starting position over time Focus Displays the residual focus error in microns after the Autofocus system has moved the objective stage over time The instantaneous error is displayed in light blue and the time based average is shown in dark blue Time Series There are two Time Series graphs Both plot the PMT volt age levels over time The red and black signals represent the IR scatter sig nals measured by each of the two PMTs Each broadening of the line represents an object Note There should be no signal above 10 volts from peak to peak this measures saturation In addition the signal should look symmetrical and consistent with previous runs with similar objects Frequency Spectrum Plots the flow speed vs signal strength as measured by the PMTs represe
92. peed CV falls consistently below 0 2 indicating that the core stream has stabilized The event rate under Acquisition status should be 100 300 beads per second If not see Troubleshooting on page 103 Flow Speed CY 0 8 5 0 6 TT D4 ep 0 2 Li 0 Fiu l l 3892 4000 4092 Observations 22 OPERATING THE IMAGESTREAM USING INSPIRE CALIBRATING AND TESTING THE IMAGESTREAM USING ASSIST Once the SpeedBeads are running and the flow speed CV is consistently less than 0 2 you can calibrate and test the instrument using the ASSIST tab For more information on the individual calibrations and tests refer to the Figure ASSIST Calibrations on page 59 in chapter 4 1 Click the ASSIST tab 2 Near the bottom of the page click Start All Note Instrument calibrations may also be run individually by selecting a particular procedure under Individual Calibrations or Individual Tests Next to each calibration or test button are the acceptable testing limits that determine whether the calibration or test passes or fails If the procedure fails the testing limit that it failed turns red If a procedure fails repeat it If it fails twice see Chapter 5 Troubleshooting or call your Amnis Field Service Representative Note that some calibrations are not routinely run with Start All 3 When the calibrations and tests have passed return to the Setup tab DATA ACQUISITION After the ImageStream system is calibrated
93. quiring data and saving it in a file When you click Run Setup the words turn grey and the Stop button is available and turns red Clicking Stop stops camera operation e Run Acquire Runs the camera and acquires image data saving it in a file When you click Run Acquire the words turn grey and the Stop button is available and turns red Clicking Stop stops camera operation e Pause Acquisition Pauses the saving of the data file until Resume is chosen at which time acquisition of the file continues Note If you click Stop before the specified number of data objects have been acquired a dialog box appears that allows you to discard the data or save it in a file Flush Lock Load Backflushes the sample and bead lines and rinses the sam ple syringe with sheath fluid three times introduces a 50 ul air bubble loads and primes the sample to deliver the proportion of beads and sample material INSPIRE SOFTWARE OVERVIEW 43 that is set in the Core Size and Speed area of the Setup tab and establishes a core Use this option when you change samples e Return Sample Returns the sample from the sample line back into the sam ple uptake port Dual Prime Rapidly delivers both the sample and SpeedBeads to the flow cell in the proportion set in the Core Size and Speed area of the Setup tab Prime Beads Rapidly delivers SpeedBeads to the flow cell Initialize Fluidics Empties the sheath and flush syringes loads new sheath f
94. r a line or a region Rgn of the imagery 46 INSPIRE SOFTWARE OVERVIEW E7 image Display Tools Region of Interest ntensiy Prolls D Pu Line Fign Mrisum 24 l Show Crave 45 Maximum 47 40 5 a l Show Migrant Took Fisel Information Mean 27 09134 Pinet 849 350 Awa ne i oJ intently 24 E 216869 aq 1 l 0 100 200 300 5 213177 Rasel fi Comectice Pixel Information box Displays the selected Pixel x y coordinates and its Intensity value Region of Interest box Displays the Minimum Maximum and Mean pixel intensity values their standard deviation Std Dev and the Area of the drawn region Intensity Profile Plot of horizontal pixel number vs Mean pixel intensity for the drawn region e Pause Stops the update of image display without stopping data acquisition The left and right arrow buttons lt lt and gt gt can be used to page up or down through the images The Pause button changes to Resume while paused Resume continues the regular update of image display pause resume e s ACQUISITION GRAPHS AND TOOLS The buttons to the left of the acquisition plots open the Cell Classification window which is used to set cell classifiers to identify objects to include in the acquiring data file and reset the dots acquired into the plots Open the cell classifier by clicking on the button cell classifier gt reset INSPIRE M SOFTWARE OVERVIEW 47 Open
95. r will decrease Bead Concentration vs Time The concentration of beads measured by both PMT channels red and black of the Autofocus Flow Speed system Used by Field Service for diagnostic purposes only INSPIRE SOFTWARE OVERVIEW 93 Setup ASSIST Advanced Setup Advanced Fluidics Autofocus Flow Speed Camera Illumination Display Acquisition FN Flow Speed Tracking Enabled IR Laser On Off Fluidics Diameter microns fs 830 nm CD 40 4 Percent Beads Foo M Camera Sync Enabled Core Velocity mm sec M Remove Background Desired fs M Flow Speed Waveform Update Bubble Vent tu ERTE fase gt O ce Closed PMT Settings Flow Speed Calculated Values Flow Speed 66 358 C PMT RelZ Mag os3 J Cy 06 Camera Sync 3342 Camera Clock Rate 145 366 5983 Signal Level Threshold 4000 Flow Speed Flow Speed Error CY 68 0 8 EE a a 0 5 4 66 an dde A 64 al 1 Ce set 1 ia 4619 4700 4819 4619 4700 4819 Time Series Volts vs Time Frequency Series 12 00 10 00 5 00 24 1 1 1 11 00 f 1 0 1000 2000 3000 4096 0 10000 15686 Time Series Volts vs Time Bead Concentration vs Time 26 l 24 li ad FA 1 y TT SR IN U T d Ai RA 7 24 1 1 1 11 54 1 1 1 0 1000 2000 3000 4096 3654 3700 3800 3854 94 INSPIRE SOFTWARE OVERVIEW CAMERA TAB The Camera tab is used to control the properties of the six channel CCD camera Note You must click Stop an action b
96. rance Officer INFORMATION AND SAFETY 3 EXPLANATION OF SYMBOLS TABLE 1 LABEL LOCATION HAZARD Waste tank Risk of exposure to transmissi ble biological disease Power supply cover Rusk of injury by electric shock Power supply Protective earth ground DANGER Visible and invisible laser Inside surface of hood Rusk of exposure to hazardous laser radiation Interior side panels near release mechanisms and next to hood latch release Rusk of exposure to hazardous laser radiation On the back of the instrument No laser radiation is accessible to the user during normal instrument operation ELECTRICAL SAFETY Equipment ratings The ImageStream is rated to the following specifications 100 240 VAC 50 60 Hz and 3A Electrical hazards are present in the system particularly in the main power supply To protect against electrical shock you must connect the instrument to a properly grounded receptacle in accordance with the electrical code that is in force in your region 4 INFORMATION AND SAFETY S CURIT ELECTRONIQUE Alimentation 100 240 V altenatif 50 60 Hz 3A Les hazards lectrique se trouvent dans l appareil surtout pr s de la source d alimentation Pour viter les choks lectriques introduire la lame le plus large de la fiche dans la borne correspondante de la prise et pousser fond LASER SAFETY The Im
97. ration and Testing e Data Acquisition Daily Shutdown Procedure Optional upgrades FLUIDICS OPERATING THE IMAGESTREAM USING INSPIRE 17 STERILIZER CLEANSER AND DEBUBBLER These recommended reagents have been formulated to optimize the performance of the ImageStream seals valves syringes and lines The use of the recommended reagents is required for proper operation of the instrument The Sterilizer Cleanser and Debubbler reagents are used in the Sterilize and Debubble scripts Reagent Name Source Catalog Cleanser Coulter Clenz Beckman 8546929 Coulter Debubbler 70 Isopropanol VWR 42101 Sheath PBS Invitrogen 14190 provided for information only other sources of the same reagent may be used WASTE FLUID The waste bottle holds all of the fluids that have been run through the ImageStream and can hold up to 1600 ml Add 160 ml of bleach to the empty waste tank It is recommended that the waste bottle contain 10 bleach when full and that the waste line remain immersed in the liquid at all times to prevent changes in the flow rate SHEATH FLUID Two bottles are provided one labeled Sheath to be filled with phosphate buffered saline PBS with no surfactants for running samples and one labeled Rinse to be filled with de ionized DI water for rinsing the instrument during shutdown Fluid is drawn from these bottles into the sheath and flush syringe
98. reased by run ning the core stream at a lower velocity and or increasing the camera gain to 10x Advanced Camera tab Core stream position is The core tracking and focus tracking objective grossly off center within positions should not change significantly from day the flow cell due to air or to day If either value is radically different 20 clog in the fluidics microns objects may rotate due to interactions with the sheath stream An off center core stream is caused by air or clogs in the fluidic system See solution for Air or clog in the sample syringes or flow cell 488 excitation laser is The laser is misaligned if the scatter control value misaligned set by ASSIST is abnormally has failed Run the Laser Power Test on the ASSIST tab again and call Amnis service if it fails a second time Everything is Image display settings are Decrease the image display gain and change to lin too bright or set too high ear in the appropriate camera channel images are red or flat in appearance 110 TROUBLESHOOTING SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Instrument sensitivity is Decrease the excitation laser power minimum is 10 set too high mW Decrease the camera stage setting minimum is 32 rows to decrease camera sensitivity in the fluores cent channel Refer to Setting ImageStream Sen sitivity guidelines Set the brightfield intensity to 800 counts The core stre
99. rked view the debris images and look for objects excluded by the cell classifiers Also observe changes to the appropriate acquisition plots after clicking the Reset button Note Any classifiers that are turned on will remain set for the subsequent sample unless they are actively turned off Coincident objects will be collected during acquisition but will be removed by IDEAS when the data file is opened by reap plying the classifiers Check all channels to be collected for compensation control files 26 OPERATING THE IMAGESTREAM UsiNnG INSPIRE COLLECTING AND SAVING THE DATA FILES 10 11 12 Once the sample is running and the ImageStream is properly set up you are ready to acquire the data as a raw image file rif This file contains uncompensated pixel data along with instrument settings and ASSIST information in a modified TIFF format The file includes only those objects that meet the current cell classifi cation criteria You also have the option of saving the objects classified as debris and beads in separate files Selup Acquistion Cak lo Acqua 5009 4 Daa Fie Filename 3e Sequence k 1 E M Save Debris Enter the number of cells you want to acquire in Cells to Acquire under Acqui sition on the Setup tab Enter the file name for the acquired data in the Data File box Note The number in the Sequence box is appended to the file name fol lowed by the rif extension The sequence number increa
100. rom the File menu optional 30 OPERATING THE IMAGESTREAMX USING INSPIRE OPTIONAL UPGRADES USING EDF Extended depth of field EDF is a novel technique used in a variety of applications including FISH spot counting where having the entire cell in optimal focus is crit ical to obtaining accurate results There are two steps to utilizing the 16 um EDF first images must be acquired with the EDF element in place and second the data must be deconvolved using the EDF kernel prior to analysis Calibration of the element is done when installed and should be repeated by Amnis service when any optical changes are made to the instrument See EDF Excitation Utility on page 82 TO COLLECT A DATA FILE USING THE EDF ELEMENT Set instrument settings for the experiment Select EDF1 from the collection filter dropdown menu Adjust cell classification settings to accomodate using EDF The calibration kernels saved during the last EDF calibration will be appended to the file and the file name will be appended with EDF Cell images as they appear on the instrument with left pair or without right pair EDF element in place Raw Max Pixel values indicated in upper left EDF image of a small bead showing characteristic L shaped pattern GENERAL CHARACTERISTICS OF USING EDF The EDF element spreads all points of light within a cellular image into consis tent L shaped patterns When EDF images are opened in ideas the data is dec
101. rough the typical operating region of the autofocus system It is used to determine each move the focus motor makes to correct focus errors The result and the limits for the calibration are shown below the list when the calibration is selected If the Multimag option is installed the instrument automatically multiplies the S curve slope by the appropriate factors to compensate for the changes in slope that result from the additional 60X and 20X magnification 80 INSPIRE SOFTWARE OVERVIEW BRIGHTFIELD CALIBRATION UTILITY The Brightfield Calibration Utility measures and stores the light output response as a function of input current for each LED used in the Brightfield illumination system In this calibration the optical system is commanded to place itself in a specified configuration 40X mag highest contrast aperture setting 60mm s flow speed 256 level staging etc The input current is varied over a predefined range and the light output is measured at the camera The resulting data are fit to a linear equation which are then used by the system rapidly set the illumination in a given channel for given configuration when requested i Brightfield Calibration Utility History 9 25 2009 10 35 47 AM Parameters Num Settings Channel Of Interest Num samples in Total Range Database Setting Saved Value Result Value INSPIRE TM SOFTWARE OVERVIEW 81 EDF EXCITATION UTILITY Extended depth of field
102. s a retro illumination scheme to maximize the amount of light incident on the cell The vast majority of light incident on the core stream passes through the stream and through cells and other particulates in the stream The retro illumination system captures this light and redirects it back on to the core stream to double to the total amount of light incident on cells in the stream In this calibration the retro reflective system is panned in manner nearly identical to the Horizontal Laser Calibration Using the same technique the optimal position of the retroreflection system is determined to maximize intensity and reduce measurement variation Retro Calibration Parameters Num Settings Channel Of Interest Num samples F Total Range 2 kA 2 a 1 1 1 1 1 1 860 840 820 800 780 760 Retro Position um Idle Database Setting Saved Value Result Value ASSIST ExcPowerT est_LaserPower_488 100 00 ASSIST RetroCalibCenterPasition 820 00 INSPIRE SOFTWARE OVERVIEW 67 ASSISTTESTS A test is a sequence of operations designed to measure the performance of a specific subsystem When a test is performed one or more test parameters are generated and evaluated against predefined limits The test results and acceptable limits are listed on the ASSIST display tab Values outside of accepted limits are highlighted with a light red background ASSIST allows complete automated operation of all tests as well as t
103. s after 45 60 minutes of run over time lines ning resulting in a drop in cell event rate Stop and save the acquisition return sample add volume to 50 vl and re run the sample after pressing Flush Lock and Load There is a clog or air bub See solution for Air or clog in the sample syringes ble in the system or flow cell Sample syringe is empty Flush sample load the next sample and dual prime TROUBLESHOOTING 109 SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Sheath syringe is empty Load sheath then dual prime Flow rate slows Sheath syringe is empty Load sheath then dual prime or stops over time There is a clog or air bub See solution for Air or clog in the sample syringes ble in the system or flow cell Leak in the fluidic system Call Amnis service Fluorescence Image display settings are Increase the image display gain and or change to imagery set too low log in the appropriate camera channel appears too dim Sample did not label well Look at the sample with a fluorescent microscope Instrument sensitivity is Increase the excitation laser power maximum for not optimized the 488 is 100 mW Increase the camera stage setting maximum is 256 rows to increase camera sensitivity in the fluores cent channel Refer to Setting ImageStream Sensi tivity guidelines If the probing protocol results in dim staining sen sitivity of the instrument can be inc
104. s and limits of the test are shown below the list when the test is selected 86 INSPIRE SOFTWARE OVERVIEW ADVANCED SETUP TAB The Advanced Setup tab contains seven additional tabs that allow access to advanced operation of the ImageStream Collectively these tabs contain all of the same controls that appear on the Setup tab plus additional controls and performance graphs that are used primarily by Amnis service engineers As a result much of the information in this section is for informational purposes only The Advanced Setup tab contains the following seven tabs e Advanced Fluidics Enables control of all fluidic components including fill ing emptying and agitating syringe pumps and moving valve positions e Autofocus Provides controls for the autofocus and core tracking systems as well as real time graphs of autofocus performance e Flow Speed Provides controls for setting and measuring the core stream velocity and synchronizing the flow speed with camera time delayed integra tion TDI line rate e Camera Enables control of the TDI camera settings including the individual sensitivity of each of the six channels and the spatial resolution of the collected images Illumination Provides control over the brightfield and fluorescence excitation laser intensity the color of the brightfield illumination and the Scatter Track ing system e Display Allows you to customize the display color and gain for each camera ch
105. s on this tab report PMT based measurements of the Autofo cus Flow Speed system that relate to Flow Speed Tracking Note Double click on a graph to view the Chart Properties window which allows you to adjust any of the graphs Flow Speed Plots the calculated core stream velocity in mm sec over time Time Series There are two Time Series graphs Both plot the PMT volt age levels over time The red and black signals represent the IR scatter sig nals measured by each of the two PMTs Each broadening of the line represents an object Note There should be no signal above 10 volts from peak to peak this measures saturation In addition the signal should look symmetrical and consistent with previous runs with similar objects Flow Speed CV Plots the calculated coefficient of variation CV of the core stream velocity over time Frequency Spectrum Plots the flow speed vs signal strength as measured by the PMTs represented in the red and black Time Series graphs The flow speed is calculated from the fast Fourier Transform of the time based PMT signals The frequency of signals received through the gratings by the PMTs from an object is related to the core stream velocity in mm sec The focal position of the core stream is calculated from the balance of signal amplitudes of the two PMTs If the core stream drifts in the z axis the amplitude measured by one PMT will increase while the amplitude mea sured by the othe
106. scom CDASF TNF B 10pg 15min B3 1031 DATE Fed C Documents and Setings amris Desktop ISK Data 043010X101 WB MONO NFkB TNF 15 5000 043010 WB CD4SFITC it arr com CD4SF TNF C 109 15min B4 Bonge DATE Fed C Documents and Settings amnis Desktop ISX Data 043010X101 WB MONO NFkB TNF 15 5000 042010 WB CDASFITC iat onris com CDASF TNF A 1009 18min 8 Highly recommended select Error notification Email from the list of Standard parameters and type in the user email address in the Apply to Selected box 9 When done click Save 10 Click Start to run the plate e A warning may be displayed if there are undefined or partially defined wells Select Yes to return to plate definition or No to continue INSPIRE Template File Not Specified Some wells have only been partially defined Wells that are not fully defined will not be included when running a plate definition Would you like to return to the definition screen to edit the plate No The Auto Sampler Unattended Operation window opens with the Plate Definition you just saved If you wish to choose a different Definition browse for it by click ing on the folder icon If you want to edit the Plate Definition click Edit This Plate and you will be taken to the Well Plate Definition window 36 OPERATING THE IMAGESTREAM UsinG INSPIRE Auto Sampler Unattended Operation elk A Open Door P Start Fr Testing 030910 X101_SF Est This Pate Edi
107. sed to check communication with the camera These settings should be used by an Amnis Field Service Representative only View Raw Frames Allows you to view images in Frame mode Select Frame in the Camera Mode box and click View Raw Frames The button name changes to Stop when you are viewing frames To return to viewing images in TDI mode click Stop select TDI in the Camera Mode box and click Run Setup 96 INSPIRE SOFTWARE OVERVIEW Setup ASSIST Advanced Setup Advanced Fluidics Autofocus Flow Speed Camera ttumination i Display Acquisition M Trigger Source m Magnification Camera Mode Automatic C 20 TDI LA ann Line Rate Hz 5000 FAN Manual C 60x Frame M Imagery Settings Bin Mode Resolution Speed Pixel Size Ref Ch Camera 1 None microns Highest Lowest 5 Ref Ch Camera 2 None al ue Higher Lower 1 C Lowe Higher 2 Lowe Highest 4 m Camera Stages rM Test Pattern gt Temperature Camera 1 31 30 256 v Off CCD 1 19 2C Primary Secondary Gains and Offsets Gain 0 1023 Offset 0 1023 Channel 1 a 179 q 86 neh oot out Pau Channel 2 175 q 86 EG PA A Channel 3 168 7 86 MO ONE 0 noir Channel 4 E 173 ci 86 OO CODE Ro Our Channel 5 175 7 86 iitinkiod roo oon oo Channel 6 a 178 J 86 tot E D OT Collection Mode Device 1 Block NDI C ED
108. ses by 1 with each suc cessive data acquisition Browse to select an existing folder or to create a new folder in which to save the files Note File names must be 256 or fewer characters in length including the path and file extension In addition file names cannot contain the following characters Naa ar ror Optional If you want to save the debris in a file check the box next to Save Debris The debris file name is appended with debris You may also save the beads in a file by checking the Save Beads box on the Advanced Acquisition tab The bead file name is appended with beads Note when you are collecting a control file turn brightfield and scatter 785 nm laser to OFF and enable collection of all channels otherwise leave bright field set to ON The names of control files that are run with no brightfield will have the suffix noBF appended to them When you are collecting a data file with EDF the file name will have the suffix EDF appended OPERATING THE IMAGESTREAM USING INSPIRE 27 13 Ensure the flow Flow Speed CV falls consistently below 0 2 indicating that the core stream has stabilized Flow Speed CY 0 8 5 0 6 TT 04 Leg 0 2 LL 0 re 3892 4000 4092 Observations 14 Acquire the data a Click Run Setup to start imaging SS isp ire for t Run Setup Displays imaging Run Acquire Begins data acquisition E Pause Acquisition Pauses the data acquisi
109. t 1945 9 Set Default 100 00 Relative gt 10 Default Reset Auto Focus Stage Position um AFStage 1 I 2400 2480 Time Series Volts vs Time I 3000 1 1 1000 2000 Time Series Volts vs Time 24 1 1 1 11 0 1000 2000 3000 4096 1935 9 Go Default tReverse Limit Focus um AF Stage vs Time Frequency Series 12 00 10 00 5 00 00 lt j 0 10000 15686 Bead Concentration vs Time 22 oH PARA it min 1 vs 0 tt 1 1 3581 3700 3781 INSPIRE SOFTWARE OVERVIEW 91 FLOW SPEED TAB To prevent streaked images the TDI camera line rate must be synchronized with the speed of objects in the core stream The ImageStream continuously and automatically adjusts the line rate to account for minute variations in core stream velocity using signals derived from the Autofocus Flow Speed system This system focuses SpeedBead IR laser side scatter light through a series of gratings onto PMTs The signal from the PMTs is processed to generate a flow speed control signal that is used to constantly update the camera line rate to maintain image synchrony The Flow Speed tab allows you to control the camera synchronization process The Flow Speed tab contains the following settings e Flow Speed Tracking Enabled When checked this box turns on the infra red IR laser and begins Flow Speed Processing FSP which results in PMT based measurements of IR laser sc
110. t This Plate dit This Plate Return sample after running each well IV Sterilize after running plate m Well Definition seka Select the defined wells that you wish to run a Load the plate into the AutoSampler and press the Start button Plate Definition sy T sjojsjojejojeiojo wl f 0 5 occ ots Acquisition Tina File Name 6_28_2010_A01 E 28_2010_404 Fe 28_2010_A05_ e 28_2010_ A06_ je 28_2010_A07 le 28_ 2010_A08 L LECLCERR __ lt Magnification gt rif 6_28 2010 402 628 2010 403 lt Magnification gt rif lt Magnification gt rif _ lt Magnification rif _ lt Magnification rif _ lt Magnification if _ lt Magnification rif _ lt Magnification gt rif Status LE 28_ 2010 4093 __ lt Magnification gt rif 628 201 0_B01 lt Magnification rif 6_28_2010_B02 _ lt Magnification rif 6 282010 B03 lt Magnification gt rif 628 2010_B04__ lt Magnification gt rif 6_28_2010_B05 ___ lt Magnification gt rif 11 Check or uncheck the boxes Return sample or Sterilize Note that these boxes may be checked or unchecked while the plate is running and the operation will apply after the current sample is finished 12 Select the wells to run they will appear in the list 13 Click Open Door to extend the plate nest 14 Place your plate on the nest w
111. t_Value 0 68 ASSIST Collection mageQualityFocusT est_VertRatio 0 88 ASSIST Collection mageQualityFocusT est_HorizRatio 0 83 ASSIST Collection mageQualityFocusT est_VertAngleT op 1 783 54 Start The test also reports regional scores which are not tested against limits The scores include the energy ratios for line profiles in the horizontal and vertical axes displayed at the bottom of the regional score grid and summed energy values for the horizontal vertical and diagonal directions radiating outward from the center of the image The summed energy values are displayed in a 3 x 3 array The value in the center of the array is the ensquared energy ratio for the single pixel in the center of the image If the MultiMag option is installed and ImageQuality Ensquared energy test will be performed for each magnification 78 INSPIRE SOFTWARE OVERVIEW ASSIST UTILITIES ASSIST Utilities are a special set of procedures that are used to enable the collection of data Unlike ASSIST Calibrations or Tests no parameters are set for later use or tested against predetermined limits The utilities are a simply a set of repeatedly used procedures that facilitate the collection of data for other alignments and tests The Utilities are not run as part of the automated Start All ASSIST commands AUTOFOCUS S CURVE UTILITY The Autofocus S Curve is the response of the Autofocus system to changes in focus There is a broad region of this curve in
112. the 12 channel options is installed the Dark Current calibration will be simultaneously performed for both cameras 62 INSPIRE SOFTWARE OVERVIEW BRIGHTFIELD CROSSTALK COEFFICIENT CALIBRATION The brightfield cross talk calibration measures the amount of spectral leakage between channels using the brightfield illuminator This calibration illuminates each channel individually and characterizes how much light leakage is present in the remaining five channels The purpose of this calibration is two fold First the spectral leakage values are used to spectrally correct the imagery in IDEAS by removing any Brightfeld light leakage from the other five channels The second purpose is to ensure that the spectral characteristics of the instrument remain constant over time The Brightfield cross talk calibration will simultaneously calibrate leakage from all eleven channels if the 12 channel option is installed in the instrument i Brightfield XTalk Coefficient Calibration History 3 25 2009 9 04 54 AM Parameters Num Settings Channel OF Interest Num samples i Total Range gt a a B a 2 Bo iS 2 E Fe p 6 1 1 35 4 Camera Channel Database Setting Saved Value Result Value ASSIST DarkCurrent0_Gen2_2 21 5039 21 ASSIST DarkCurrentl_Gen2_2 21 5625 21 ASSIST DarkCurrent _Gen2_2 22 168 22 ASSIST DarkCurrent3_Gen2_2 23 082 22 ASSIST BFCrossT alkCoefficientsChant 1 00 0 01 0 ASSIST BFCrossT alkCoefficie
113. the list of classifiers menu with a right click of the mouse in the cell classifier window Select the features to use from the list Ignore All Enabled allows you to quickly toggle the enabled features on and off to observe the effect of the classifiers You can set upper and lower limits for the following features in any of the channel images Es Cell Classifier Parameters SEE M Channels to Collect Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 N Collected M Collected M Collected M Collected M Collected M Collected Area Lower Limit Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Area Upper Limit Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 gO CE CE i Oo ou Show All Enabled Hide All Ignore All Enabled Area Lower Limit Area Upper Limit Maximum Gradient Intensity Lower Limit Maximum Gradient Intensity Upper Limit Mean Intensity Lower Limit Mean Intensity Upper Limit Minimum Intensity Lower Limit Minimum Intensity Upper Limit Peak Intensity Lower Limit Peak Intensity Upper Limit Percent Saturated Pixels Lower Limit Percent Saturated Pixels Upper Limit Total Intensity Lower Limit Total Intensity Upper Limit Total Saturated Pixels Lower Limit Total Saturated Pixels Upper Limit You can use the Cell Classifier window to set cell classifiers to identify objects for inclusion in or exclusion from the acquiring d
114. the segmentation mask algorithms critical for image feature calcu lations you should use a 1X camera gain Camera Mode The camera is normally run in TDI mode which prevents image streaking or in Frame mode which does not To view images in Frame mode select Frame and click View Raw Frames at the bottom of the tab To return to viewing images in TDI mode press the Stop button at the bottom of the tab change back to TDI mode and click Run Setup Trigger Source The camera line rate can be controlled by the Autofocus Flow Speed system Automatic mode or by the camera line rate in Hertz in Manual mode The camera line rate can be entered in the Line Rate field Manual mode should be used by an Amnis Field Service Representative only Collection Mode Controls a six position filter wheel that is in the optical path immediately before the spectral decomposition element INSPIRE SOFTWARE OVERVIEW 95 Open has no element 658 405 Block ND 2 0 and 3 0 are Neutral Density Filters used for ASSIST tests EDF Element is a wavefront coded extended depth of field element used for producing an extended depth of field while collecting imagery Note Instruments with optional lasers have the additional 658 and or 405 block ing elements in this position to enable the optional lasers with EDF Blocked has a blocking element used for certain ASSIST calibrations e Test Modes The two options generate test patterns that are u
115. tion until Resume is chosen at which time the file continues to be collected Stop Stops either Run Setup or Run Acquire geStream Vie _ir rumert Sample Lesds Help D Pauses image display while images are still being acquired View images previously captured b Click Run Acquire to collect a data file The Flow Speed Amplitude graph displays only the history of the signal strength instead of the real time frequency magnitude spectrum while acquisition is taking place 15 Follow the progress of acquisition under Acquisition Status The acquiring data can be viewed graphically on the Acquisition Plots at the bottom of the window 16 The data file s are automatically saved in the selected folder once the desired num ber of objects are collected To prematurely stop acquisition click Stop The system prompts you to either discard the acquired data or to save the collected data in a file The acquisition can be paused and resumed 17 Enter the file name destination and the number of events to acquire 18 Click Run Acquire to collect and save the first experiment data file 19 Once acquisition finishes click Flush Lock Load to collect the next sample The sample lines will be flushed and sheath will be sent through the sample uptake port 50 ul of air will be drawn into the sample port to prevent dilution of the sam ple with sheath Note If the next sample has no nuclear dye and follows a DNA intercalating dye stained sampl
116. tive stage to default focus and core positions Toggle the IR laser off and then on by clearing then checking the FSP Enabled box System has not been primed Prime beads or dual prime Pumps are not running Prime beads or dual prime Pumps are empty If sheath syringe is empty load sheath then dual prime If the bead pump is empty load beads then dual prime Bead concentration is too low Make sure percent beads is set to 10 The fluid in the bead pump should be cloudy If it is clear then beads did not load properly Under the Beads menu choose Flush and Load Flow rate is too slow or fast Open the bubble trap Advanced Flow Speed tab for a few seconds to slow flow rate down rap idly Press Lock on to attain proper flow speed Find the core stream by manually panning the objective Turn Autofocus tracking off With the laser at 100 mW track the core left and right with 10 um relative movements When laterally cen tered manually track focus If the core stream is found outside its normal Core and Focus Tracking setting then there may be a clog or air bubble in the fluidic system Air bubble in the flow cell 104 TROUBLESHOOTING If you suspect bubbles in the flow cell poor or erratic core formation run the Purge Bubbles script from the Instrument dropdown menu If that does not work return your sample and run the Sterilize script SYMPTOM
117. tter information gathered by the Autofocus Flow speed optical system to track the optimal focal position of the core stream Laser side scatter information is collected on two separate PMTs that are focused on opposite sides of the core stream in the z axis When the PMT signal amplitudes are equal to one another the imaging objective is focused on the center of the core stream When the core stream moves out of focus the PMT signal amplitudes become unbalanced This information is used by the autofocus system to continuously move the objective in the z axis to achieve optimal focus during a sample run The following sections of the Autofocus tab provide manual control of the objective z axis position as well as control over the AutoFocus system Flow Speed Tracking Enabled When checked turns on the infrared IR laser and begins Flow Speed Processing FSP which allows PMT based mea surements of IR laser scatter off of the SpeedBeads e AutoFocus Tracking Enabled When checked enables the automatic objec tive stage movements to the best focus position as determined by infrared IR signals collected by the PMTs e AutoFocus Enabled Turns the autofocus system on e AutoFocus Error Notification Indicates that the stage has moved out of range e PMT Settings Displays the PMT settings critical to optimal performance of the Autofocus Flow Speed systems Do not change these values e Focus Tracking Allows you to move the objecti
118. und intensity values are measured and the thresh olds are set based on these measurements Default Thresholds Resets the threshold values to 0 Note Changing the display properties does not change the data They are for dis play purposes only SQUELCHING DEBRIS Some samples have an abundance of small particulate debris These can be eliminated from collection by using Cell Classifiers or by using Squelch to reduce the sensitivity of object detection As opposed to classifying debris away from cells squelching debris can prevent INSPIRE crashes related to overburdening the computer processor with an abnormally high event rate Squelch should only be used if the rate of total objects per second reaches Squelch values range from 0 to 100 increasing the value decreases object detection sensitivity Turn off all cell classifiers Observe the relative proportion of cell to debris images appearing in the imaging area and the event rate Total Sec under Acquisition Status On the Advanced Setup Acquisition tab increase the Squelch value until the observed proportion of cells to debris increases in the imaging area Observe the Total Sec event rate on the Setup tab under Acquisition Status If it is still greater than 500 repeat step 2 29 OPERATING THE IMAGESTREAM USING INSPIRE SETTING IMAGESTREAM SPEED AND RESOLUTION The optimal operating speed is set at the factory for each instrument and is approximately 66 mm s
119. up Tab Stop Stops the three syringe pumps Bubble Vent Controls the Bubble Vent Valve Opening the valve depressurizes the fluidic system by venting the bubble trap to air through the standpipe You can rapidly drop the core stream speed by opening this valve e Flow Speed Calculated Values Flow Speed Displays the current calculated core stream velocity in mm sec 92 INSPIRE SOFTWARE OVERVIEW CV Displays the current calculated variation in the core stream velocity as a percentage of the mean sample speed coefficient of variation CV Camera Clock Rate Displays the current calculated TDI clock rate in Hertz of the camera This value is used to synchronize the camera with the speed of the objects in the core stream PMT Rel Z Mag A parameter that compensates camera synchronization for object size This value should be edited by an Amnis Field Service Rep resentative only Camera Sync Sets the relative magnification of the optical system The accuracy of this number is critical to keep the camera synchronized with the speed of objects in the core stream Click Calibrate to automatically calcu late and set this value Signal Level Threshold Sets the PMT signal threshold for the Frequency Spectrum graph Only PMT signals above the level threshold will be con sidered by the Autofocus Flow Speed system when calculating focus posi tion and core stream velocity Graphs All six graph
120. utton on the left side of the screen to stop camera operation before changing these settings You can use and adjust the following settings on the Camera tab Sensitivity Allows you to specify for each channel the number of camera pixel rows or stages over which to integrate the signal The greater the stage number the greater the sensitivity Typically all non scatter channels are run at identical stage settings while the scatter channel channel 1 is run at 32 or 128 stages All of the channels can be set to the same stage settings by clicking Syn chronize Channels Note The relationship between camera stage setting and sensitivity is not linear For example a channel run at 128 stages will be nearly 20 fold less sensitive than the same channel run at 512 stages Bin Mode Determines the camera pixel size Increasing the pixel bin size allows the camera to synchronize with higher core stream velocities which increases sample throughput However increasing the bin size also decreases image spatial resolution The bin mode should always be set to Highest until further testing has been done with the other settings RESOLUTION SPEED PIXEL SIZE MICRONS Highest Lowest 0 5 Higher Lower 1 0 Lower Higher 2 0 Lowest Highest 4 0 Gain Allows for a camera gain setting of 10X which increases camera sensitiv ity but also increases background noise Because the increased background noise affects
121. ve in the x axis relative to the core stream The lateral position of the core stream in relation to the objective lens is determined by camera based measurements of object x centroid This infor mation is used by the Automatic Core Tracking system to continuously move the objective to the optimal lateral position during a sample run The following sections of the Autofocus tab allow you to control the objective z axis position and the Automatic Core Tracking system e Automatic Core Tracking Includes parameters for the Automatic Lateral Core Tracking control loop The Min Time between Updates Core Tol erance and Filter Length values should only be edited by an Amnis Field Service Representative only e Core Tracking Allows you to move the objective in the x axis so that object images are collected in the center of the camera channels All values are expressed microns Absolute You can enter an x axis position in this field Click the nght arrow button to move the objective stage to this position Relative Distance that the objective stage will be moved in the x axis when the right or left arrows are clicked You can use this feature to posi tion the objective to achieve optimal focus on the core stream Reset Returns the objective to the x axis position specified in the Default window Current Displays the current position of the objective stage in the x axis Click Set as Default to reset the default position to this p
122. ve in the z axis to achieve optimal focus on objects in the core stream All values are expressed in microns Absolute You can enter a z axis position in this field Click the right arrow button to move the objective stage to this position 88 INSPIRE SOFTWARE OVERVIEW Relative Distance that the objective stage will move in the z axis when the right or left arrows are clicked You can use this feature to position the objective to achieve optimal focus on the core stream Reset Returns the objective to the z axis position specified in the Default window Current Displays the current position of the objective stage in the z axis Click Set as Default to reset the default position to this position Default Displays the default position of the objective stage in the z axis Click Go to Default to move the objective to this position At Reverse Limit Turns green when the objective stage reaches the limit of its mechanical range of motion in the z axis e Focus Offsets Includes parameters for the Autofocus control loop Caution The A K B and Hyst Comp fields are pre set to optimize the move ment of the objective in response to movement in the core stream These fields should be edited by an Amnis Field Service Representative only e Autofocus Calibrate Launches an ASSIST test that should be run by an Amnis Field Service Representative only Core Tracking is accomplished by moving the objecti
123. ve your settings as a template for future use Template file names are appended with the suffix ist They are saved in the INSPIRE ImageStream Data folder Load Default Template Use this template to calibrate the instrument Exit and Shutdown Instrument Turns off the instrument control system and exits INSPIRE Exit Exits INSPIRE Instrument menu Run the ImageStream camera and instrument specific fluidic scripts automated fluidic routines Instrument Run Setup FS Run Acquire F6 Load Sheath Load Flush Syringe Initialize Fluidics Sterilze System Purge Bubbles Dual Prime Service Scripts Run Setup Allows you to view the imagery and adjust the instrument set tings without collecting data Run Acquire Acquires image data and stores it in a file Load Sheath Fills the sheath syringe with sheath fluid and an air bubble that facilitates stable flow Load Flush Syringe Fills the flush syringe with sheath fluid Initialize Fluidics Empties the sheath and flush syringes loads new sheath fluid and flushes the instrument with the new sheath fluid The sam ple and the SpeedBeads are also flushed out of their respective pumps The sample uptake line is loaded with 50 ul of air in preparation for a sample load SpeedBeads are loaded into the bead pump and are primed Speed Beads will be running at the end of this script Sterilize System Decontaminates and cleans the ImageStr
124. viously worked Advanced Flow Speed tab Core stream position is grossly off center within the flow cell due to air or clog in the fluidics The core tracking and focus tracking objective positions should not change significantly from day to day If either value is radically different 10 microns the core may experience poor laminar flow resulting in high flow speed variations and streaked imagery An off center core stream is caused by air or clogs in the fluidic system See solution for Air or clog in the sample syringes or flow cell TROUBLESHOOTING 107 SYMPTOM POSSIBLE CAUSES RECOMMENDED SOLUTIONS Excessive flow speed vari See solution for Air or clog in the sample syringes ation due to air or clogin or flow cell system Insufficient fluid volume Fill the waste tank with enough fluid to immerse in the waste tank the waste line outlet Cells are not Lateral deviation of the See solution for Air or clog in the sample syringes centered in the core stream due to air or or flow cell channel clog in the system Core stream is Core and Focus Tracking Reset the core and focus tracking motors and go to not in the nor values are incorrect the default position mal core or focus tracking range Deviation in the core See solution for Air or clog in the sample syringes position due to air orclog or flow cell in the system Autofocus is not tracking Reset the focus position If the cells
125. y 2 0 40 Channel Df Interest r Focus Position um Focus Position um INSPIRE SOFTWARE OVERVIEW 85 S CURVE PEAKS UTILITY Measures the depth of modulation of the AFFS S curve The autofocus system provides an error signal that proportional to defocus in microns When the error is 0 the system is in the best focus position The error signal theoretically modulates between 0 9 and 0 9 with a linear error response centered around best focus As objects are moved continuously away from focus in either direction the S curve will peak with a negative or positive value at positions 7 5 microns from best focus After this point the error signal will begin to decrease and eventually drop off to 0 again The S curve peaks test pans through 15 microns of focus and measures the peak response in the negative and positive directions The peak responses are tested against limits to ensure that the focus system provides enough modulation depth to pull objects into focus The positive and negative peak values are stored in the ASSIST database ES PMT Focus Utility Parameters Num Settings 40 o Channel Of Interest _ Num samples 5 m Total Range 40 104 8 n 1 PMT Energy 0 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 Focus Position um PMT 2 10 Pe 7 1 1 1 1 1 1 1 1 1 D 0 1 2 3 4 5 6 7 8 3 10 6 PMT Energy 4 2 0 Focus Position um The result

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