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Imagestream Inspire Manual

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1. 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 62 Focus Offset Beads 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 Focus Offset Beads Test Parameters Number of Settings 15 Channel of Interest Number of Samples 500 Total Range 15 E E o E o o a Database Setting Saved Value R
2. Ch 1 Delta from Mean Optimal Position Ch 2 Delta from Mean Optimal Position Ch 3 Delta from Mean Optimal Position Ch 4 Delta from Mean Optimal Position Ch 5 Delta from Mean Optimal Position Ch 6 Delta from Mean Optimal Position 65 Chapter 4 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 the 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
3. 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 47 Chapter 4 Spatial Offsets Calibration Measures and stores 12 calibration factors for the vertical and horizontal registration of each spectral channel 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 ImageStreamXmust maintain sub pixel spatial registry between channels amp Spatial Offsets 40x Calibration elc ES Parameters Number of Settings 1 Channel of Interest Number of Samples 1000 Total Range 1 Continuous Mode Database Setting Saved Value Result Value ASSIST XOffsets Gen2 0 246 03 ASSIST SpatialOfsets Min 40 950 ASSIST SpatialOffsets Max 0 950 ASSIST SpatialOffsets2ndRefMin 3 000 ASSIST SpatialOffsets2ndRef Max 3 000 Ch1 X Offset Stat 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 be
4. top 296 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 296 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 66 amp Image Quality 40x Ch 6 Test Parameters Number of Settings Channel of Interest Number of Samples Total Range Focus Position vs Ensquared Energy 0 70373 0 64 05 04 4 Ensquared Energy 0 34 0 23761 T 4 3 5 3 Focus Position um Database Setting Saved Value Result Value ASSIST CollectionlmageQualityFocusTest LimitMin 0 550 ASSIST CollectionimageQualityFocus Test Limit Max 1 000 ASSIST CollectionimageQualityFocus Test_PercentUsed 0 020 ASSIST CollectionlmageQualityFocusTest Value 0 754 sbre Chapter 4 EE Image Quality 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
5. 36 button 23 92 SpeadBeads run 45 Speed control 22 35 SpeedBeads loading 28 Startup 28 button 23 Sterilizer 14 Task bar information 23 Templates 31 Test BF Intensity Selection 57 BF Uniformity 58 Camera Noise 59 Excitation Laser Power 56 Flow Core Axial Stability 60 Flow Core Lateral Stability 61 Flow Core Position 62 Focus Offset 63 Focus Percentage 64 Focus Uniformity 65 Image Quality 66 Tests 56 Troubleshooting 69 93 Chapter 6 user name 28 waste 5 9 bottle 14 fluid 14 Weight 2 94
6. Opens the compensation wizard Load Matrix Applies compensation to the Intensity features View Matrix Opens the compensation matrix values table Clear Matrix Stops applying compensation of Intensity features Layout menu Layout Vertical Horizontal Auto resize Anslysis Area Vertical View the image gallery and analysis area side by side Horizontal View the image gallery and analysis area top and bottom Auto resize Analysis Area When selected automaticaly adjusts the sep arator between the image gallery and analysis area when images are added or removed from the view Advanced menu For field service personnel only 26 Fluidics Advanced Fluidics Autofocus Flow Speed Camera Illumination Acquisition Autosampler Error About ImageStream Access the current INSPIRE version number with the About ImageStream option Help About amnis part of EMD Millipore INSPIRE for the ISX mkII 27 Chapter 3 Daily Operations Turning on the ImageStream 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 tum the ImageStream on if the power is off Note If the ImageStream power is on and INSPIRE is already launched go directly to step 4 1 Press the green power button inside the front door of the ImageStream to turn on the
7. Template Loads factory settings Generate RIF file Check to save a Raw Image File during acquisition Generate FCS file Check to save a Flow Cytometry Standard file during acquisition Exit and Shutdown Instrument Turns off the instrument control system exits INSPIRE and shuts down Exit Exits INSPIRE Instrument menu Run the ImageStreamX camera and instrument specific fluidic scripts automated fluidic routines Instrument Calibrate with ASSIST Load Sheath Flush Load Beads Load Flush Syringe Prime Purge Bubbles Purge Sample Load Line View Tank Levels Service Scripts Options Calibrate with ASSIST Opens the Calibrations and Tests window Load Sheath Fills the sheath syringe with sheath fluid and an air bubble that facilitates stable flow 24 Fluidics Flush Load Beads Flushes the bead syringe and reloads beads from the bead tube Load Flush Syringe Fills the flush syringe with sheath fluid Prime Pushes sample and beads into the flow cell 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 rinsing the flow cell The sheath syringe is then refilled with sheath and the bubble trap lines and flow cell are filled with sheath Purge Sample Load Line Flushes the sample load line with debubbler to remove bubbles formed during sample loading View Tank Levels O
8. To immediately turn the machine off should the need arise remove the plug from the socket outlet 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 1096 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 personnel 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 d
9. nawa mama E fore Remamwemme 00 E peser _rewanovaregononascatiot 18 Fluidics The Instrument Control Panel The instrument control panel provides tools to control instrument operation data acquisition and status Sample _ Volume 0 0 uL Time 0 00 All 0 obj s l Filename default noBF 1 rif Fg Show All Custom Filename Text Seq defaut a wa Collect 1000 E of Al 405 2000 Ejmw Comme ba 12 488 poo mw prighifeid 642 150005 mw OFF 785 o 50 E mw Setintensity 4 m Magnification 20X 40X 60X Lo Speed Hi Le M Hi Sensitivity Lo Focus Centering La 4 v 9 m gt H 19 Chapter 3 20 Fluidics Sample 2 pi In the Sample section you can load a sam ple or return a sample Sample time remaining is displayed when Sample Time Remaining 5 34 a sample is running Acquisition NR q In the Acquisition section you can run in O lu setup mode begin acquisition pause or mM stop acquisition The Filename and the population and the rate ofthe population Filename 060211 F5101 PKH26 Comp Control being collected is displayed All 1593 obj s ind File Acquisition Custom Filename Text 060712 X197 MII Ftc In the File Acquisition section you can type in a custom filename set the sequence choose the data file folder type the number of events and c
10. 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 Chapter 1 S curit Laser L ImageStreamX c est une appareil au laser Classe 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 lasers ImageStreamX peut avoir les lasers suivants Longueur opna a Puissance Maximale 815 840 nm 180 mW Les etiquettes d avertissement suivantes sont place s dans l interior du capot DANGER Visible et invisible radiations laser quand ouverte et les enclechements sont defaltes Exposition dangereuse au rayonnement direct ou diffus pour loail 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 Biological Safety Biological Safety Biohazards The Image Stream is rated at BSL1 Do not load or flush samples containing infectious ag
11. 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 ina 3x 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 68 Chapter 5 Troubleshooting This section is designed to help you troubleshoot the operation of the ImageS treamX Mark II If additional assistance is required contact the Amnis service department System Unstable fluidics Air or clog in system e Fluidics respond sluggishly Event rate slows overtime Event rate is slower than expected e Cross contamination from previous samples Erroneous fluid level indicator Instrument will not pass ASSIST Compensation wizard fails to complete Software INSPIRE appears to freeze NSPIRE Fails to launch Plots fail to update or update slowly Datafile fails to collect Image Noimages e Imaging and acquisition rate is erratic or appears frozen Objects appear streaked Objects are not centered in the channel Objects are rotating in the core stream Objects are out of focus or distorted Objects are cropped The two brightfield images are not of the same cell Images appear pixelated or larger than normal Objects appear larger or sma
12. running e Verify the channel for compensation Collect the All population if all are positive or draw a region on the Uncom pensated Intensity scatter plot to define the positive population View the pop ulation in the Image Gallery and choose this population to collect Name the file and choose the path to save the data Click Collect File The compensation coefficients are calculated The com pensation coefficients and an Intensity scatter plot using the coefficients are displayed Click OK on the Acquisition Complete popup window e Click Load to continue with the next single color control sample or click Retum optional 33 Chapter 3 e Repeat the previous steps for each compensation sample For each sample reset the Image gallery population to view All and then create an appropriate population for each sample Note the R1 gate can be moved for each sample Click Exit when done and Save the coefficients to a compensation matrix file The template is restored and the saved matrix is used to compensate the Inten sity feature Note that imagery and other features are not compensated The com pensation can be cleared if desired from the compensation menu Scatter plots can be made with the feature Uncompensated Intensity to compare with and with out compensation 19 Continue to collect experimental files 20 Click Shutdown when done See Daily Shutdown Procedure 34 Data Acquisition Optional se
13. set tings are set too low Insufficient illu mination Core stream posi tion is grossly off center within the flow cell due to air or clog in the fluid Excitation laser is misaligned tings are set too high Instrument sen Increase the image display gain by clicking on the channel column select ing the appropriate channel and mov ing the right green handle bar to a smaller value or the brightest pixel in the histogram To set the display back ground to black move the left green handle bar to the dimmest pixel in the histogram Tum the appropriate lasers on Set the laser powers to maximum and decrease them to prevent pixel sat uration If the probing protocol results in dim staining sensitivity of the instrument can be increased by changing the fluidics speed to Lo Hi sensitivity mode Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics Run calibration particles on the Flow Sight Load the default template Open ASSIST re run the laser alignment calibration for the appropriate laser line and verify it passes Decrease the image display gain by moving the right hand green handle bar to a higher value for the appro priate camera channel Decrease the excitation laser power to prevent pixel saturation Saturation is indicated in the image gallery by pix els colored in a contrasting color gen 83 Chapter 5 urates while the others d
14. that may be chosen as a group or individually See the list of parameters abover Check or uncheck the desired parameters The user can also define custom parameters Expand the category to see the individual parameters To delete a custom parameter select it and use the delete key Click OK when done Toinclude a parameter in the file name click in the box below the column heading make sure it says yes Columns can be re ordered by click drag 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 Select Defined button or e All In this example column 1 is selected 40 Optional upgrades ES Well Plate Definition y start Plate Definition Name E ae Customize Well Plate Select by Color EF B Bm pg EE Select Defined Delete Selected Wells E Ex IAE IE CCCCCCE eeeeeee Ov 6600606666 CC 000 60066666 e e6666 696 96 96 66066666 666 69 96 96 6 CCC Ole eeeeeee Ole eeeeeee Of TZEREN 7 You can edit values for some of the Custom and many of the Standard param eters You can do this for all selected wells or for individual wells 8 When done click Save 9 Click Start to run the plate Awarning may be displayed if there are unde
15. the X Axis at 0 allows you to set the display pixel intensity to 0 for all intensities that appear to the left of that line Doing so removes background noise from the image Click dragging the vertical green line on the right side allows you to set the dis play pixel intensity to 255 for all intensities that appear to the right of that line Note Changing the display properties does not change the pixel intensity data They are for display purposes only Select Channel Ch01 Enable Minimum Pixel Intensity Maximum Pixel Intensity Channel Names Colors Cl Ch02 Ch03 Ch04 Ch05 Ch06 Ch07 Ch08 Ch089 Ch10 Ch11 Ch12 d oJ 0 Automatic Manual Image Display Mapping X Axis Scale Set to Default Full Scale amp s S S S S S S S S S Set Range to Pixel Data Autoscale Set Linear Curve Reset All Settings Reset to White Saturation Color m Reset to Default Colors Enable All Channels a J 17 Chapter 3 The Analysis Area Graphs are displayed in the analysis area during setup or acquisition Regions can be drawn on the graphs to create populations The functionality of the anal ysis area is the same as in IDEAS Refer to the IDEAS user manual for further information on graphs regions and populations Analysis Area Tools SWE httt5 BEe amp ss O Ll Icon Name Deseipton n pesa aana
16. 00 mb using the right and left arrows xm Runs the shutdown script and sterilizes the system Bottom task bar Status buttons are displayed at the bottom of the INSPIRE window Switch Core Mode completed at 8 40 AM on 6 6 2012 Flush Lock and Load Sample running Describes the current scri pt Acquiring 060612 X197 60X LPS 2 nf Click this button to abort a script Level indicator for pumps Green indicates compensation is being applied to the Intensity feature Note that imagery and other features are not compensated Yellow calibrations and tests not run Compensation 3 Red one or more calibrations or tests ASSIST failed Green all calibrations and tests have passed 23 Chapter 3 Menu Bar The menu bar is located in the upper left portion of the INSPIRE screen It consists of these four menus 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 File New Template Load Template TUDE Seve Template Load Default Template Generate RIF file Generate FCS file Exit and Shutdown Instrument Exit New Template Create a new template Load Template Browse for and open saved templates Save Template Save your settings as a template for future use Tem plate file names are appended with the suffix ist They are saved in the INSPIRE Data folder Load Default
17. 2 ASSIST BFCrossT alkCoefficientsChan3 Channel Of Interest 1 1 35 4 Camera Channel Saved Value Result Value 21 5039 21 21 5625 21 22 168 22 23 082 22 1 00 0 01 0 0 06 1 00 0 0 07 0 04 1 50 Core Stage Position Calibration The alignment of the stage in the X direction is controlled so that the position of the core is centered in the field This calibration finds the core position using the X centroid position of the SpeedBeads and calculates an offset from the factory setting and sets the position of the stage in the X dimension Core Stage Position Calibration Parameters Number of Settings 1 Channel of Interest Number of Samples Total Range Core Stage Position Offsets 36 07 gt 30 4 20 4 104 0 E 5 E g n o E 5 bi a 104 21 35 1 D 0 200 400 600 800 1000 Sample Number Idle Database Setting Saved Value Result Value Mean Offset from Current Position ASSIST CoreStagePositionOffset 2 779 51 Chapter 4 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 med
18. Autofocus and cen tering is not tracking properly Verify brightfield is working normally and rerun ASSIST using calibration beads See solutions for unstable fluid ics Verify the beads will run by returning any sample going to fluidics section and press stop then run Next go to the advanced drop down select flow speed and check that the red and black histograms have tight CVs at the appropriate core velocity To view bead images select the All population and check include beads Verify that there are two brightfield channels Check in the image display properties that 1 and 9 are active ver ify that brightfield is emitting in chan nels 1 and 9 Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics In the Focus and Centering section adjust focus and centering left or right until the images are centered and in optimal focus 80 Symptom Objects are rotat ing in the core stream Objects are out of Possible Causes Recommended Solutions The core tracking and focus tracking should not change significantly from Core stream posi day to day If either value changes rad tion is grossly off ically objects may rotate due to inter center within the actions with the sheath An off center flow cell due to air core stream is caused by air or clogs or clog in the fluid Jin the fluidic system Run the purge ics bubbles script from the instrument drop do
19. DEAS 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 11 Chapter 2 Technology Overview 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 5000 objects per second The time delay integration TDI detection technology used by the ImageStreamX 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 ImageStreamX works detector dichroic filter stack m P velocity detector PP YA wv autofocus celis in flow E 4 brightfield illuminator Hydrodynamically focused cells are trans illuminated by a brightfield light source and orthogonally by laser s A high numerical aperture NA objective lens collects fluorescence emissions scattered and transmitted light from the cells The co
20. INSPIRE 9 ImageStreamX System Software User s Manual Version Mark Il January 2013 For updates log in at www amnis com Patents and Trademarks Amnis technologies and products are protected under one or more of the fol lowing 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 7610942 7634125 7634126 7719598 7889263 7925069 8005314 8009189 8103080 8131053 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 EMD Millipore 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 Part of EMD Millipore 645 Elliott Avenue W Suite 100 Seattle WA 98119 Phone 206 374 7000 Toll free 800 730 7147 WWwW amnis com Contents Patents and Trademarks 44 anneau ii DISCIAIMEYS e ee Oe ae ee lt n ii Technical ASSIS nes sse tod ied ded a EEE ii O ONES corto A
21. Position 830 000 lllumination RetroHysterisis 10 000 54 Autosampler Nest Calibration The ImageStream autosampler runs a self calibration This calibration verifies that the sipper can self calibrate and find the home position If the calibration fails or is not run the autosampler will not run Autosampler Nest Calibration Parameters Number of Settings 2 Channel of Interest Number of Samples Total Range Idle Database Setting Saved Value Result Value 55 Chapter 4 ASSIST Tests 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 the 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 collec
22. See solu variation due to air levels tions for unstable fluidics Light source deliv Power down and power up the instru ering variable out ment if this does not fix the problem put call Amnis service Brightfield intensity Intensity set before Allow the system to stabilize after level sets incor desired flow speed loading a sample and then click Set rectly has been achieved Intensity Power down and power up the instru ment if this does not fix the problem call Amnis service Movable optics are out of position 85 Index Acquisition button 21 stop 21 Acquisition button 21 Analysis Area 18 tools 18 ASSIST 45 ASSIST Calibrations 47 Autosampler Nest 55 Brightfield crosstalk coefficient 50 camera synchronization 47 Core Stage Position 51 Dark Current 49 Horizontal laser 52 retro 54 side scatter 53 spatial offsets 48 ASSIST Tests 56 BF Intensity Selection 57 BF uniformity 58 Camera Noise 59 Excitation laser power 56 87 Chapter 6 flow core axial stability 60 flow core lateral stability 61 flow core position 62 focus offset 63 focus percentage 64 focus uniformity 65 image quality ensquared energy test 66 Autosampler 38 Brightfield adjustments 22 31 Calibration 28 45 Autosampler Nest 55 Brightfield Crosstalk Coefficient 50 Camera Synchronization 47 Core Stage Position 51 Dark Current 49 Horizontal Laser 52 Retro 54 Side Scatter 53 Spatial Offsets 48 Calibra
23. age 45 To collect a data file using the EDF element 1 2 3 4 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 EDF image of a small bead Cell images as they appear on the instrument with left pair or showing characteristic L without right pair EDF element in place Raw Max Pixel values shaped pattern indicated in upper left General characteristics of using EDF The EDF element spreads all points of light within a cellular image into con sistent L shaped patterns When EDF images are opened in ideas the data is deconvolved to create an image of the entire cell projected simultaneously in focus During acquisition and before deconvolution images will appear blurred into characteristic L shaped pattems and raw max pixel values will be lower with EDF than with standard mode collection Compensation controls for EDF data can be collected with or without the EDF element in place 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 15100 instrument or 4095 for the ISX As long as the images did not saturate the camera during acquisition th
24. and Tests 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 synchronization helps ensure crisp image collection Z Camera Synchronization 40x Calibration bl R Parameters Number of Settings 13 Channel of Interest 4 Number of Samples 500 Total Range 4 Camera Sync 1st Camera Sync Grad Metric Camera Sync Camera Sync 2nd Camera Sync Grad Metric T T T 38 5 39 395 Camera Sync Database Setting Saved Value Result Value ASSIST CameraSyncStart 39 000 ASSIST CameraMagMin 37 100 ASSIST CameraMagMax 42 000 ASSIST CameraMag 38 053 1st Camera 2nd Camera Start M a 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
25. 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 may be 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 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 75 ul samples to the 96 well plate cover with Sigma Aldrich X Pierce Film XP 100 Cat 2722502 and load the plate into the autosampler Runthe plate see procedure below Access to AutoSampler operations is found under the AutoSampler menu 38 Optional upgrades Autosampler Eject Tray Load Tray Define Plate Run Plate Load From Well From this menu you may Extend or retract the tray Create a plate definition Runaplate Runasingle well from a plate To begin 1 Choose Define Plate from the Autosampler menu to open the Well Plate Def inition window Start El Add Remove Wel
26. ate Third party sheath buffers cannot be used With the system powered down look for Fluid lines are leak leaking sheath Verify the fluid lines ing SpeedBeads fail to run mount snuggly into position Call Amnis service Verify the beads will run by returning any sample going to fluidics section and press stop then run Next go to the advanced drop down select flow speed and check that the red and black his tograms have tight CVs at the appro priate core velocity To view bead images select the All population and check include beads 71 Chapter 5 Symptom Possible Causes Recommended Solutions The sheath syringe should contain 2 4 ml of air to buffer the movement of the pump s microstepper motor If too little air is present run the start up script With the system powered down look for Fluid lines are leak leaking sheath Verify the fluid lines ing mount snuggly into position Call Amnis service Cells settle in the lines after 45 60 min utes of running resulting in a drop in cell event rate Stop and save the acquisition Return the remaining sample restore the sample volume to 30ul and re load the sample to continue acquisition Data can then be appended together in IDEAS There is a clog or Run the purge bubbles script from the air bubble in the sysjinstrument drop down menu See solu tem tions for unstable fluidics amplie synnge 15 Load a fresh sample empty Sheath syringe is Load
27. ation Guide for experimental set up recommendations Use compatible sample solutions from the table below Sample order Samples from an experiment are typically run in the following order Experimental sample with the brightest stains to set the sensitivity for the run Single color DNA dye control NO BF or SSC to ensure correct dye con centration 10 bleach to wash out DNA dye followed by PBS Single color fluorescence controls no DNA dye NO BF or SSC Therest of the experimental samples with DNA dye Note compensation controls may be collected after experimental files if desired Loading and running the sample 1 Press Load and load an aliquot of the brightest sample in the experiment that fluoresces with each fluorochrome used It is critical that you run this sample first to establish the instrument settings DO NOT change laser settings for the exper iment once established on this sample if you are using dyes that are excited by more than one laser When prompted place sample vial with 20 200 ul into the sample loader 30 2 Data Acquisition In the file menu choose Load 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 appropriateness of the settings for the specific experimental run Choose t
28. atter Cal ibration failure Laser Power test failure Brightfield align ment test failure mm be a fluidics issue see the Flow rate stops or slows over time section Tests may fail if the system is reloading sheath or failed to set up properly Re run the test by clicking in the box next to the test and pressing the start button in the popup window If the test fails three times in a row call Amnis service Verify brightfield is working properly Verify brightfield is working properly Verify brightfield is working properly Make sure the excitation lasers are off and brightfield is blocked Completely power down the instrument and power back up to re run the test Brightfield XTalk caljVerify brightfield is working properly and ibration failure that spatial offsets passed Verify the laser turns on and can set power properly Completely power down the instrument and power back up to re run the test Verify spatial offsets passed Verify the laser turns on and can set power properly Verify spatial offsets and frame offsets passed Verify the 785 SSC laser turns on and can set power properly Completely power down the instrument and power back up to re run the test Verify spatial offsets passed Verify the laser turns on and can set power properly Completely power down the instrument and power back up to re run the test Verify spatial offsets and frame offsets passed Verify brightfield is working p
29. ay Optimizing instrument setup for sample runs is also described here in detail e Fluidics INSPIRE User Interface Daily Operations Data Acquisition Daily Shutdown Procedure Optional upgrades 13 Chapter 3 Fluidics 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 JSoucer Catalogs Cleanser Coulter Clenz amp Packman 8546929 Coulter Debubbler 7096 Isopropanol wR M2101 O 0 DAO Ta Hypo VWR JT9416 1 chlorite She Bs mien 4190 Rise deionized war 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 1096 bleach when full 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 dra
30. curacy 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 Brightfield Uniformity Test u a F Parameters Number of Settings 12 Channel of Interest Number of Samples Total Range Pixel Intensity Channel BF Uniformity No Gains Corrections 2 7 2 7e x a More Charts Database Setting Saved Value Result Value CV for Ch 1 Corrected Uncorrected CV for Ch 2 Corrected Uncorrected CV for Ch 3 Corrected Uncorrected CV for Ch 4 Corrected Uncorrected CV for Ch 5 Corrected Uncorrected CV for Ch 6 Corrected Uncorrected CV for Ch 7 Corrected Uncorrected Start 58 Camera Noise Test The electronic noise is measured with no illumination to the CCD in two successive frames The fluctuation is measured on a pixel by pixel basis amp Camera Noise Test Parameters Number of Settings 2 Channel of Interest Number of Samples Total Range Database Setting Saved Value Result Value ASSIST CameraNoiseResults 1456 14 ASSIST CameraNoise UpperLimit 3 000 59 Chapter 4 Flow Core Axial Stability Test Measures the stabilit
31. e 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 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 Laser Safety The ImageStream 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 1 Wavelength Maximum Power 370 380 nm 30 or 85 mW 400 413 nm 150 mW 483 493 nm 200 mW or 400 mW high power option 558 562 nm 200 mW The following laser warning label appears on the inside surface of the hood ADANGER Visible and or invisible Laser Radiation Avoid direct exposure to beam The following laser warning label appears on the interior side panels near release mechanisms and
32. e amount of total intensity is collected Image collection speed is inversely related to image resolution sensitivity mI sensitivity Lo 35 Chapter 3 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 Fillthe Rinse Cleanser Sterilizer and Debubbler bottles if necessary 2 Empty the Waste bottle 3 Remove any tubes from the uptake ports 4 Click Shutdown 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 con tamination It takes about 45 minutes of unattended walk away operation to complete 36 Optional upgrades Optional upgrades Using EDF Extended depth of field EDF is a novel technique used in a variety of appli cations 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 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 Blue ASSIST Tab on p
33. e most contaminating sample cells or run the sterilize script 30min Tank has moved Open the buffer compartment and move away from the sen the tank closer to the sensor until the fluid sor level indicator is correct Power down and power up the instru Sensoris broken ment if this does not fix the problem call Amnis service Cells are not dis played due to over clipping DNA dye from pre vious sample is labeling current sample Incorrect template Go to the file drop down and select load loaded default template Re run ASSIST Verify the beads will run by returning any sample going to fluidics section and press stop then run Next go to the advanced drop down select flow speed and check that the red and black his tograms have tight CVs at the appro priate core velocity To view bead images select the All population and check include beads The particles must be running gt 1000 SpeedBeadsare events per second and without sig not running properly nificant clumping If the beads are diluted or clumped try running a fresh tube of beads If the problem persists there may SpeedBeads fail to run 73 Chapter 5 Symptom P S iii Calibration and or test failure Focus adjustor cal ibration failure Frame Offset cal ibration failure Spatial Offsets cal ibration failure Dark Current cal ibration failure Horizontal Laser Calibration failure Retro Calibration failure Side Sc
34. eck or uncheck the boxes Return samples Sterilize and Shutdown 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 11 Select the wells to run they will appear in the list 12 Click Eject Tray to extend the plate tray 13 Place your plate on the tray with well A1 positioned at the upper left corner 14 Click Start to begin 15 The Status column will be updated for each well as it is run For each sample the instrument performs the following in sequence 1 Load 2 Validation flow speed CV focus brightfield intensity object rate 3 Data Acquisition 4 Result success or error 16 During a run 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 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 def inition Acquisition will pause until the user intervenes f an 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 42 Optional upgrades f the same error occurs on three consecutive wells the autosampler aborts the plate and sterilizes the instrument if the Sterilize after running plate box
35. eld Crosstalk Coefficient Calibration 50 Core Stage Position Calibration 51 Horizontal Laser Calibrations 52 Side Scatter Calibration 53 Retro alia aa 54 Autosampler Nest Calibration 2 2 2 55 ASSIST TESIS so lata cada a na ue Ido tela oid caet eee c ee 56 Excitation Laser Power Tests 0 ooo oo occ oe cece cece cece cece neces 56 BF Intensity Selection Test 57 BRUNO EOS oret ee tite 58 Cam ra NolSB Mestre sun the ins Roda ecd 59 Flow Core Axial Stability Test 60 Flow Core Lateral Stability Test 61 Flow Core Position Test LR 62 Focus Offset Beads TeSt use e dte hassan esse 63 Focus Percentage Test s sese sse 64 Focus Uniformity FES ee a aan rae Maa aee tl Aide des a aii Det 65 Image Quality Ensquared Energy Test 66 VS LE ce eere ays Selec Es Aleman ney LTDA aa E Mani 22 I LEE Ra RR E 69 SO BNIIB cic eed e i ieu LLL IL ea D LL ede EL Lund 69 VVC ass rn E CATAR AR RIP SISI MUA ce DR la a 69 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 t
36. elected 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 16 Fluidics Setting the Image Display Properties 1 Clickon in the acquisition section to open the window 2 Select the channel by clicking on the channel name 3 Tochange the channel name type a new name To change the channel color click on the color box 4 Tosetthe display mapping adjust the right and left green bars in the graph You will adjust the Display Intensity settings on the graph the Y Axis to the Pixel Intensity the X axis The range of pixel intensities is 0 4095 counts The display range is 0 255 The pixel intensities shown in gray are gathered from the images coming through in the specific channel and updates with every 10 images Updates to the adjustments can be visualized in the image gallery At each intensity on the X Axis of the graph the gray histogram shows the number of pixels in the image This histogram provides you with a general sense of the range of pixel intensities in the image The dotted green line maps the pixel intensities to the display intensities which are in the 0 255 range Manual setting is done by Click dragging the vertical green line on the left side crossing
37. ents without first exposing the sample to inactivating conditions It is recommended that samples be fixed in 296 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 the 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 S curit BiologiqueBiorisques 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 est recommand que les chantillons soient fix s dans du paraformald hyde 296 pendant au moins 10 minutes avant d acqu rir des chantillons avec l ImageStreamX 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
38. ese pixel values are valid Object Morphology and System Masks will be smaller in EDF mode 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 37 Chapter 3 EDF images exhibit increased texture due to higher resolution Brightfield imagery is not as crisp in EDF mode as in standard mode Anin depth discussion of EDF can be found in the following reference Cytom etry 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 cardiomyocytes or epithelial cells The pixel size using 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 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 Using the Autosampler To enable high throughput experiments and unattended operation the autosampler option includes upgraded fluidics software and an imbedded tray for loading of samples in a 96 well plate format Prior to running the plate a plate definition is created that
39. esso A Lc STR Ne A On AU OD We E iii General Information and Safety 2 Declaration of Conformity aaria oaao ao iroa 111n 4 Explanation of Symbols mm mm amaan mm mmama mmama 5 Electrical Safety een 6 S curit Electronique 6 Laser Safety eee 7 S curit LASER coo eres reel Ree Ui t rte 8 Biological Safety eee 9 S curit BiologiqueBiorisques 9 Spare Parts List 9 Technology Overview 12 PUIG Sie ng RIR UR cte oL vaL A t INR DA 14 Sterilizer Cleanser and Debubbler 14 ANS ONU OS Ue Pp TT EER CR 14 Sheath Fluid es ttr sent rer A estate 14 INSPIRE User Interface odo ss Sot E Rs 15 The Image Gallery ue de ceo su creel E Rm e dt 16 Image Gallery Tools ee 16 Image Display TOONS 4 eo e A aoe a AORN Me v edat 16 Setting the Image Display Properties 17 The Analysis Areas oce Wa AA A AA ei ere 18 Analysis Area TOOIS 221i Monet sa aca Laetare toon urbaine setae uon 18 The Instrument Control Panel 2 020 000 ooo cence eens 19 Men Bat sc fecal ente fofa Sub cm TAO Eita Su Jobe vd duo YA NESI edere oa lear eS a 24 Daily Operations seren
40. esult Value Mean Focus Offset Mean Focus Offset Camera 1 Mean Focus Offset Camera 2 Ch 1 Delta from Mean Actual Offset Ch 2 Delta from Mean Actual Offset Ch 3 Delta from Mean Actual Offset Ch 4 Delta from Mean Actual Offset Start 63 Chapter 4 Focus Percentage Test Measures the percentage of SpeedBeads in focus and sets a limit of 90 Focus Percentage Test Parameters Number of Settings 1 Channel of Interest Number of Samples 3000 Total Range z o o B o a o 5 9 Lu o N o 50 Gradient RMS Idle Database Setting Saved Value Result Value Poca Fos ASSIST FocusTestPercentLowerLimit 90 000 ASSIST FocusTestGradient Threshold 55 000 64 Focus Uniformity Test Measures the best focus position for every channel and then calculates the difference of each channel from the mean for all channels The tolerance for focus uniformity ensures that all channels are in optimal focus Focus Uniformity Test Parameters Number of Settings 20 Channel of Interest Number of Samples 100 Total Range 30 Channel 1 NI 18 406 1 1 I I 1 1 i 24 20 15 10 5 0 527 Focus Position um Gradient RMS Channel 2 Gradient RMS 1 1 1 1 1 1 1 li 24 20 15 10 5 0 bay Focus Position um More Charts Idle Database Setting Saved Value Result Value Mean Optimal Focus Position
41. et up you are ready to acquire the data as a raw image file rif and or an FCS file The rif con tains uncompensated pixel data along with instrument settings and ASSIST infor mation in a modified TIFF format The file includes only those objects defined by the population selected in the acquisition section LI File Acquisition Custom Filename Text Seq 060712 X197 Mil Fc 1 E gis Collect 1000 of A Enter the number of cells you want to acquire after Collect and select the pop ulation To add another population click the box Enter the file name The number in the Sequence box is appended to the file name followed by the rif extension The sequence number increases by 1 with each successive data acquisition Files collected with BF off will be appended with noBF and files col lected with EDF enabled will be appended with EDF in the file names 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 lt gt or 10 Browse to select an existing folder or to create a new folder in which to save the files 11 Set the Image Display Properties See Image Display Tools for more information 12 Acquire the data 32 Data Acquisition Acquisition All 1593 obj s Filename 060211 FS101 PKH26 Comp Control a Imaging should be running if not Click gt to start imaging b Click tocol
42. fined 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 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 44 Chapter 3 Eject Tray D Start Plate Definition Plate Settings test E Edit V Return samples Sterilize Shutdown aja Im 3 4 5 le 7 8 9 10 11 12 Well Definition mui DODO DD OOC Select the defined wells that you wish to run Load the plate into the AutoSampler and press the Start button 819800000000 0 0 c9 0 V DVD 00 0e o 90 9 9 9 9 9 9 9 9 092 29 EO UU ro 9o 9 9 9 9 9 9 9 9 9 9 5e CCC oo o9 9 9 9 9 9 9909209 wa fonlon File Name Status AO ide A2 A02 rf Idle A3 ADIn Idle A4 ADA rif Idle A5 ADS nf Idle AG ADE nf Idle A7 AD7 rif Idle A8 ADB nif Idle Ag ADI rif Idle A10 AlO nf Idle A11 AVL af Idle A12 AZ Idle B1 BOT rif Idle B2 BO2 rf Idle Be A 10 Ch
43. formance 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 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 Optional Click Run Beads to begin running beads without starting calibrations ortests 3 Torun one calibration or test click on an individual calibration or test and click Run 4 Tostopacalibration 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 Note Calibrations and tests do not run in order 40X Calibrations are completed before changing magnifications to run 20X and 60X calibrations 45 Chapter 4 Calibrations Spatial Offsets 60x Calibration Dark Current Calibration Tests Test 405nm Laser Power Test 9 19 2001283731AM c 488nm Laser Power Test 9 19 2012 8 37 38 AM Brightfield XTalk Coefficient Calibratio
44. he objective under Magnification option Magnification EDF 20X 40X 60X Select EDF collection if desired See Using EDF for details Turn on each laser used in the experiment by clicking on the wavelength Set the laser powers so each fluorochrome has Raw Max Pixel Intensities between 100 and 4000 counts as measured in scatterplots or histograms of the appropriate channels and there is no saturation Select the channels to be collected in the Image Display Properties by clicking Channels in the Acquisition section The default saturation color can also be set in this window See the section below for Setting the Image Display Properties Le Illumination mjo nni Gute E 2 200 00 gt mw Brightfield rm A88 642 15000 mw OFF x 785 500 i mw Setintensity Select Brightfield channels Default is Ch1 fora 6 channel system Ch1 and 9 for a 12 channel system Click Set Intensity Create graphs to gate on cells of interest Recommended Scatterplot of Area versus Aspect Ratio of Brightfield to gate on cells and eliminate debris Scatterplots or Histograms of Intensity for channels used in the experiment Scatterplots of Raw Max Pixel to observe any saturation To identify objects for inclusion in or exclusion from the acquiring data file the fol lowing features in any channel are available Area The number of pixels in an image reported in square microns Aspect Ratio The Minor Axis d
45. his information readily available for all users The safety information consists of the following areas General Information and Safety Explanation of Symbols Electrical Safety Laser Safety Biological Safety Chapter 1 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 2012 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 8096 for temperatures up to 319C with the maximum relative humidity decreasing linearly to 5096 at 409C The main s supply may not fluctuate more than 10 and must meet transient over voltage category Il 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
46. hoose the ren population to collect ustom Filename Text Type the filename Seqt Choose the beginning sequence number Navigate to the folder to save the data Enter the number of events to collect Choose the population to collect Add a second population to collect Collect 1000 21 Chapter 3 amp Illumination In the Illumination section you can turn 205 2000 FE uu laser and brightfield illumination on or off and set intensities Select the scatter chan nel either 6 or 12 All lasers have var iable power and are defined by their uie 5o ijo sims excitation bandwidth OFF and 0 mW of power ON at 60 mW of power 642nm laser excitation currently set to ON at 150 mW of power 785nm laser excitation currently set to OFF at 5 72 mW of power This laser is for side scatter only Brightfield Brightfield illumination is shown as ON in land9 yy channels 1 and 9 488 20000 7 mw Brightfield 642 15000 mw OFF Set intensity Sets the Intensity of the brightfield to 800 counts Q Magnification amp EDF Magnification Select the magnification Note this is optional equipment 40X Adjust the speed and sensitivity for the run Speed and Sensitivity are inversely a related Sensitivity Med speed is 2X binned Hi speed is 4X binned 22 Fluidics Focus and Centering Focus Centering Focus and Centering can be adjusted 02 gt 4
47. i 28 Turning on the ImageStreamX 2 28 Preparing to run and calibrating the ImageStreamxX 28 Data Acquisition oe od ecco ap ie Eae RA nt ne aa 30 Sample OO ess Nm D SHE he edes 30 Loading and running the sample 0 0 0 2 0 0 ccceceececeecececcececeeees 30 Collecting and saving the data files 32 Optional settings soon aie dn andas Es t obit Tac de aetas Adao isa Ra nar om Rea han 35 Sguelching DebHlSs u cott uos sai oat encre o DER A rent cite 35 Setting ImageStreamX Speed and Sensitivity 35 Daily Shutdown Procedure 36 Optional upgrades e uei ao S dut hates ebore te Bode eue 37 sing EIE sete RA Shetek RR A NA WA Ree ELA 37 To collect a data file using the EDF element 37 Using MultiMag iud Nh hala Lec Sat Aaah asl cat LU LED E Mare d 38 Using the Autosambpler eee ue eas aeu Seca so eae ea erect tedden da Reden ne pans 38 ODG maes ar ecoa oo td na Led 39 ASSIS BRE o MER tes a S 45 ASSIST Calibrations eto citerior Nile LA hie ed LLL LEA LAE 47 Camera Synchronization Calibration 47 Spatial Offsets Calibration 2 22 2 2 48 Dark Current GaliDFallOPi ete tueur Eo eae Ends das e etas an Ste aru 49 Brightfi
48. ian 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 488 Horizontal Laser Calibration Parameters Num Settings Channel Of Interest 1 Num samples i Total Range 488 Laser gt a n E E i 1 1 j 0 20 40 50 Horizontal Laser Position um Idle Database Setting Saved Value Result Value ASSIST ExcHorizontalLaserPosition 488 9 22 ASSIST ExcPowerTest LaserPower 488 100 00 The result for the calibration are shown below the list when the calibration is selected 52 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 amp Side Scatter 40x Ch 6 Calibration Parameter
49. if or fcs file Go to the file drop down menu and was created check Generate rif and or fcs file 77 Chapter 5 78 Image Gamera is not man ci Run Setup ER ees Stop to stop the camera and already running then click Run Setup Imaging is paused Click Resume Displayed region is In the cell view area select the all pop incorrect ulation Tum the appropriate lasers on Set the Insufficient illu 785 SSC laser to 40mw Set the laser mination powers to maximum and decrease them to prevent pixel saturation Make sure the sample concentration is between 107 and 108 cells ml Lower concentrations can be used but this will decrease the cells second Region being In the cell view area select the all pop viewed has few or ulation or readjust regions to include more cells Turn the appropriate lasers on Set the Insufficient illu 785 SSC laser to 40mw Set the laser mination powers to maximum and decrease them to prevent pixel saturation The process of object detection can safely handle up to 4000 objects per second The maximum sample con centration is 4 5x10 8 cells per ml with the recommended concentration 1 10 x10 7 cells per ml To decrease the event rate dilute the sample The sample is too concentrated 79 Chapter 5 Objects are not centered in the channel Camera is not track ing the cell velocity Lateral deviation of the core stream due to air or clog in the system
50. igital device pursuant to part 15 of the FCC rules These limits General Information and Safety 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 Chapter 1 Declaration of Conformity DECLARATION OF CONFORMIIY MANUFACTURER MODEL NUMBER REPORT DIRECTIVES STANDARDS IN ACCORDANCE TO ISO IEC GUIDE 67 FORA Cellular Analyzer Amnis Corporation 645 Elliott Avenue W Suite 100 Seattle WA 98119 Phone 206 576 6865 ImageStream X Mk II 582624 02 and F2LQ3326 02S EMC Directive 2004 108 EC amp Low Voltage Directive 2006 95 EC 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 Characteristics Limits and Methods of Measurement CISPR 11 2009 edition Radio Disturbance Characteristics Limits and Methods of Measurement EN 55022 1998 CISPR 22 1997 edition Electromagnetic Compatibili
51. instrument and start the computer Log on with the user name Amnis and password is 100 Launch the INSPIRE software and by double clicking the INSPIRE icon on the desktop Preparing to run and calibrating the ImageStream 4 Fillthe 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 com patibility chart to choose the appropriate Sheath fluid Cells in PBS run with water sheath will swell 5 Empty the waste tank Push on the quick disconnect buttons to remove the tub ing from the waste tank Add 160 ml of bleach to the Waste bottle The final vol ume of waste when full will be approximately 1600 ml and therefore the final bleach concentration for a full waste tank will be 1096 bleach It is recommended that the waste be emptied every day and fresh bleach added before Startup Click Startup 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 Click Start All Calibrations and Tests in the calibration window Center the core stream images if necessary by laterally moving the objec
52. is checked 17 A well plate report txt file 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 man ually or completed the entire plate 18 If Batching was included in the well plate definition the data files will be proc essed using the IDEAS compensation matrix and templates designated All of the cif daf and statistics report txt files will be saved to the designated output file path M Chapter 4 Instrument Calibrations and Tests ASSIST Tab ASSIST Automated Suite of Systemwide ImageStreamXTests 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 per
53. ivided by the Major Axis is a measure of how round or oblong an object is See below for the definitions for Major and Minor Axis 31 Chapter 3 Background Mean The average pixel intensity of the background pixels Gradient RMS The average 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 Major Axis The longest dimension of an ellipse of best fit Mean Pixel The average pixel intensity in an image background subtracted Minor Axis The shortest dimension of an ellipse of best fit Object Number The serial number of an object RawCentroid X The center of the object in the X dimension of the frame Raw Centroid Y The center of the object in the Y dimension of the frame Raw Max Pixel The intensity value of the brightest pixel in an image no back ground subtraction e Raw Min Pixel The intensity value of the dimmest pixel in an image no back ground subtraction Time The object s time value in seconds Uncompensated Intensity The integrated intensity of the entire object image the sum of all pixel intensities in an image background subtracted See the IDEAS User Manual for more details on features and graphing Collecting and saving the data files Once the sample is running and the ImageStream is properly s
54. 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 vis des microbes Spare Parts List The instrument contains no serviceable parts Only Amnis trained technicians are allowed to repair maintain and set up the alignment of the laser beams Chapter 2 Introduction to the ImageStream The Amnis ImageStream is a bench top multispectral imaging flow cytometer designed for the acquisition of up to 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 or within cells or between cells in cell conjugates The Amnis ImageStreamX system includes the ImageStreamX multispectral imaging flow cytometer and the INSPIRETM and I
55. l Parameters Ou Max Acquistion INSPIRE ErorNotficaton Mnmum Validate Remove B DUIPITEURCEEDCSAUNDC NEG LEE c 3 NET ET E Include in filename no no no no no Apply to selected ATE Gray 45 15 yes yes Al AD T 45 15 yes yes no Selectby Color E HEM a 10216 TE 45 15 yes yes Select Defined Wells Delete Selected Wells A3 moga date A03 Gr 45 15 yes yes M A04 T 45 15 yes yes no Au 1 C2 C C Jos Ce E C Jo 9 n2 De ma ds T m A o 9 9 9 A6 QA er DATE Gra 4 15 yes yes AT A075 T 45 15 yes yes AAAH 9 999 9 99 9 9 As Juana os E 5 ver ya clooooo o0020200920929 As nor spare a Gy 45 15 yes yes O A10 mr T G 45 5 yes yes 2 3 LJ 3 o 3 3 o 3 o 3 o 9 An TAN T 45 15 ves ves no OAEI O az wiza roare ar Gm 4 15 ves ves BI BOT T G 45 15 yes yes E 999999999999 B2 B02 T 45 15 yes yes o 5 9 0909909000 00 B3 Boat sort 45 15 yes yes eoccccoo000000020 B4 oat DATE 45 15 yes yes BS ROS nf T 45 15 yes yes o B6 poe DATE 45 15 yes yes B7 B074 DATE 807 ram 45 15 yes yes required for complete well definition Save Cancel 2 Begin to create a new definition or you may browse for a previously saved def inition to edit by clicking on the folder icon 3 Name the plate definition 4 Ata minimum each well requires an Output File Path Max Acquisition Time and Template File in order to be considered defined Other parameters can optionally be added to the definition in the next step 5 Choose the paramete
56. lect a data file 13 The data file s are automatically saved in the selected folder once the desired number of objects are collected To prematurely stop acquisition click B The system prompts you to either dis card the acquired data or to save the collected data in a file The acquisition can be paused and resumed by clicking 14 Once acquisition finishes either load the next sample or return the remaining sample Note If the next sample has no nuclear dye and follows a DNA intercalating dye stained sample Load a solution of 1096 bleach and then Load PBS to ensure that residual dye does not stain the subsequent samples 15 Change the file name for the next sample and continue collecting samples 16 Repeat for each sample 17 When finished running the experiment samples or after setting the template run single color compensation controls with the same laser settings as the exper imental samples with the exception of the scatter laser 785 which turns off in compensation mode 18 Click La in the analysis tools to begin compensation mode This turns brightfield and scatter 785 nm laser OFF and enables every channel to be collected Keep all laser powers the same as for the exper imental samples Follow the prompts in the wizard to collect all compensation control files Click Load or if a compensation control sample is already running click Next Place the tube on the uptake port and Click OK Click Next when sample is
57. llected 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 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 12 Chapter 3 Operating the ImageStream Using INSPIRETM 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 to prepare for use the following d
58. ller than normal e Notall 12 channels are being displayed mages have incorrect colors Intensity Fluorescence imagery appears too dim Fluorescence is too bright images have a contrasting color or appear flat 69 Chapter 5 One channel saturates while the others do not e Scatter is too dim or bright or changes over time Large variation in brightfield intensity levels Brightfield intensity level sets incorrectly 70 Air bubbles in fluid lines Clog in fluid lines Sample is too con centrated Inappropriate sheath solution imended Solutions Make sure a sufficient sample volume is used To clear the air bubble Run the purge bubbles script Detergents and foaming agents such as FBS can cause bubbles to form in the lines If these buffers are causing air in the system remove them from the sample and resuspend in dPBS Run the purge bubbles script Run the sterilize script followed by the startup script Load calibration beads and verify the system runs normally Filter the sample with a 70um nylon cell strainer Run the sterilize script followed by the startup script Load calibration beads and verify the system runs nor mally Clumpy and viscous samples cause cav itation in the fluidic lines and create bub bles Dilute the sample to 1x10 7 cells ml and strain the cells through a 70um nylon mesh Run the purge bub bles script Verify the sheath is dPBS De gas the sheath as appropri
59. m all test conditions The result and the limits for the calibration are shown below the list when the calibration is selected If the 12 channel options is installed the Dark Current calibration will be simultaneously performed for both cameras 49 Chapter 4 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 Brightfield 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 Brightfield XTalk Coefficient Calibration History 8 25 2008 9 04 54 AM Parameters Num Settings Num samples Total Range gt a a B i gt a z B2 E 2 5 E c by Database Setting ASSIST DarkCurrent0_Gen2_2 ASSIST DarkCurrentl Gen2 2 ASSIST DarkCurrent2 Gen2 2 ASSIST DarkCurrent3 Gen2 2 ASSIST BFCrossT alkCoefficientsChan ASSIST BFCrossT alkCoefficientsChan
60. n 9 19 2012 10 45 45 AM 642nm Laser Power Test 9 19 2012 8 37 43 AM Core Stage Position Calibration 9 19 2012 10 45 50 AM 785nm Ch 6 Laser Power Test 9 19 2012 8 37 48 AM 405nm Horizontal Laser Calibration 9 19 2012 10 46 21 AM 785nm Ch12 Laser Power Test 9 19 2012 8 37 52 AM 488nm Horizontal Laser Calibration 19 19 2012 10 46 43 AM Brightfield Alignment Test 9 19 2012 8 38 23 AM 642nm Horizontal Laser Calibration 19 19 2012 10 47 05 AM Brightfield Uniformity Test 9 19 2012 8 38 47 AM 785m Ch 6 Horizontal Laser Calibration 9 19 2012 10 47 24 AM Camera Noise Test 9 19 2012 8 38 50 AM 785nm Ch12 Horizontal Laser Calibration E 19 2012 10 47 42 AM Flow Core Axial Stability Test 9 19 2012 8 39 05 AM Side Scatter 20x Ch 6 Calibration 9 19 2012 8 45 13 AM Flow Core Lateral Stability Test 9 19 2012 8 39 14 AM Side Scatter 40x Ch 6 Calibration 9 19 2012 10 48 01 AM Flow Core Position Test 9 19 2012 8 39 20 AM Side Scatter 60x Ch 6 Calibration 9 19 2012 8 42 42 AM Focus Offset Beads Test 9 19 2012 8 40 09 AM Side Scatter 20x Ch12 Calibration 9 19 2012 8 45 24 AM Focus Percentage Test 9 19 2012 8 40 19 AM Side Scatter 40x Ch12 Calibration 9 19 2012 10 48 10 AM Focu
61. n and try launching ment INSPIRE 76 being over used Too many plots in For optimal plot update rates limit the the template number of plots to 15 Right click on the plot select graph prop erties and change the selected pop ulation to all or a population that has qualifying events Plots are scaled In the plot tool bar press the mag incorrectly nifying glass and rescale the plot Make sure there are events going into the collection region by viewing that region in the image gallery and updat ing the acquisition collection population appropriately Verify the cell concentration is appro priate 1x10 7 cells ml is ideal Verify the computer hard drive has suf ficient room to save the data file To do this go to Start Computer right click on properties and a pie chart showing how much disk space is available is dis played Backup and delete data to free Parent population has no qualifying events Data file fails to No events qualify collect for the region Computer hard drive is full Some samples have high con centrations and acquire faster than the display rate Check the destination folder and see if the raw data was col Collecting data over a downed network or changing the name of the destination folder will cause the instrument to lose the data directory Verify the data des tination folder is accessible using the browse button in the Acquisition Set tings section No r
62. ns and Voltage Variations Immunity Test EN 61000 4 11 2 ition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EN 61010 1 2008 2 edition AUTHORIZED REPRESENTATVE Izasa S A TEST FACILITY Aragon 90 08015 Barcelona Spain VAT ESA28114742 Contact Jose Maria Alonso 34 916 630 550 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 etes Ho Date 23 August 2012 Title Quality Assurance Officer Explanation of Symbols Explanation of Symbols Table 1 Lab or amd Risk of exposure to trans Waste tank missible biological dis ease Risk of injury by electric shock Power supply Protective earth ground Visible and or invisible Laser Radiation Avoid direct exposure Power supply cover Risk of exposure to haz Inside surface of hood a ardous laser radiation Interior side panels near Risk of exposure to haz release mechanisms and Lie ardous laser radiation next to hood latch release No laser radiation is acces On the back of the instru sible to the user during nor ment mal instrument operation Chapter 1 Electrical Safety Equipment ratings Th
63. o not Instrument sen sitivity is not opti Probing protocol requires better stain Instrument is expe riencing large tem Set the brightfield intensity to 800 counts by pressing Set Intensity Load sheath then go to the instrument drop down and run prime tions for unstable fluidics The best instrument setup maximizes the dynamic range of fluorescence sig nal while at the same time avoiding image pixel saturation which cannot be compensated In general decreas ing the laser powers until no pixels sat urate Reduce the concentration of the stain that produces the saturating signal so that all probes can be simultaneously imaged without excessive saturation Some DNA dyes are required to run with the sample to stain properly how ever if too much dye is in solution it can cause the core stream to flu cells can be imaged properly Typ ically the concentrations in Current Protocols in Cytometry should work Allow the instrument to warm up by running for 15 minutes Direct a fan toward the back of the instrument to dissipate excess heat or move the system to a temperature con trolled environment Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics 84 Possible Causes flow cell due to air or clog in the fluid ics Large variation in brightfield intensity Run the purge bubbles script from the Large low Speed instrument drop down menu
64. ocity 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 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 60 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 Flow Core Lateral Stability Test Parameters Number of Settings 1 Channel of Interest Number of Samples 3000 Total Range 1 8 5 2 E 2 a E o 8 0 X Centroid um Database Setting Saved Value Result Value Mean Position ASSIST LateralPositionStdDevTestValue 6 846 ASSIST LateralPositionStdDevTestMax 15 000 ASSIST LateralPositionMeanLimit 15 000 Contaminated sheath obstructions air or improper pump function may broaden the co
65. pens the fluid level window Service Scripts For field service personnel only Options File Acquisition Email Notification Automated Batching E Prompt for file location on acquire Autosampler menu Access autosampler controls Autosampler Eject Tray Load Tray Define Plate Run Plate Load From Well Eject Tray Opens the door of the autosampler and extends the tray for the 96 well plate Load Tray Retracts the plate tray back into the instrument and closes the door Define Plate Opens the plate definition dialog Run Plate Starts the autosampler run as defined by the plate definition Load From Well Allows a single sample load from a well plate 25 Chapter 3 Analysis menu Access the Feature Population and Region Managers Func tionality is the same as for IDEAS Refertothe IDEAS user manual for more information Analyses Features Populations Regions Features Opens the Feature Manager Features can be renamed or new combined features can be created Populations Opens the Populations Manager View edit or delete pop ulations Regions Opens the Regions Manager View edit or delete regions Note See IDEAS User manual for more information Compensation menu View edit or create a new compensation matrix Compensation ki Create Matrix Load Matrix View Matrix Edit Matrix Clear Matrix Create Matrix
66. re 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 61 Chapter 4 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 Flow Core Position Test Parameters Num Settings Channel Of Interest Num samples In Total Range lE I 1911s psp pepe opo o p s Bop f De ee pe ppp f alley Restoring Instrument Settings Database Setting Saved Value Result Value ASSIST FlowCorePositionT est FocusDeltaM ax 15 00 Calculated Focus Position Delta ASSIST FlowCorePositionT est FocusPosValue 219 16
67. roperly 74 Symptom Possible Causes Recommended Solutions Em Brightfield uni RUP formity test failure Verify brightfield is working properly Verify camera can image properly Com pletely power down the instrument and power back up to re run the test Verify the reagent buffers are full Run Flow Core Axial Stajthe sterilize script followed by the startup bility test failure script and re run the test See solutions for unstable fluidics Ree solution above Flow core Axial sta Stability test failure bility test test failure bility test test failure ility test aie lee solution above Flow core Axial sta test failure ility test Camera noise test failure Image Quality test See solution above Flow core Axial sta failure bility test Compensation The region to col In the wizard verify that 1 000 of All wizard fails to lectwas setincor cells or of a region drawn on the appro complete rectly priate population are being collected ee Ma ess Set the events to acquire less than 1 000 are being collected Make sure that the compensation control sample has more than 10 positive Cells are not flu events and are as bright as possible IgG orescent capture beads or a cell line stained with a single fluorochrome may be used for comp controls Compensation controls must be a sam ple with a single fluorochrome label in a single tube Each fluorochrome must be run separately Cell
68. rs you would like to use Click Add Remove Well Parameters to choose the parameters you want to report for the wells 39 Chapter 3 Add or Remove Columns ea Add or Remove Columns Add or Remove Columns 4 Date Date Custom Parameters V Well v Well ED llumination Parameters V Color V Color F BF Channel Output File Path V Output File Path 405 Power V Max Acquisition Time min V Max Acquisition Time min 17405 SSC Blocker V INSPIRE Template V INSPIRE Template 488 Power Error Notification Email 7 Error Notification Email 488 SSC Blocker V Minimum Volume uL Minimum Volume uL 561 Power V Validate Sample V Validate Sample 561 SSC Blocker 7 Remove Beads IV Remove Beads 71594 Power Comments Comments 11594 SSC Blocker Batch Batch 642 Power Dose Dose 642 SSC Blocker Time Time 71785 Power Custom Parameters Custom Parameters 785 SSC Blocker 6 llumination Parameters t lllumination Parameters Imaging Parameters amp amp lmaging Parameters G E maging Parameters E Binning Magnification Enter custom column name here Enter custom column name here Enter custom column name here Add Custom Column Delete Add Custom Column Delete Add Custom Column ok Cancel OK Cancel There are several categories of parameters
69. s Number of Settings 20 Channel of Interest Number of Samples 200 Total Range 10 2 7 E s 1 25 Power mW Idle Database Setting Saved Value Result Value ASSIST SideScatterLaserintensityMin 13 000 000 ASSIST SideScatterLaserintensityMax 16 000 000 ASSIST SideScatterLaserintensity Target 14 500 000 ASSIST Side ScatterCalib Intensity 14 362 391 ASSIST SideScatterCalibPower 1 016 55 Chapter 4 Retro Calibration The ImageStream uses 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 amp Retro Calibration Parameters Number of Settings Channel of Interest Number of Samples Total Range 2 7 5 E 5 E I 840 820 Retro Position um Database Setting Saved Value Result Value ASSIST RetroPosition 252 325 ER ASSIST ExcPowerTest LaserPower Laser2 200 000 ASSIST RetroCalibCenter
70. s Uniformity Test 9 19 2012 8 40 42 AM Side Scatter 60x Ch12 Calibration 9 19 2012 8 42 54 AM Image Quality 20x Ch 6 Test 9 19 2012 8 46 19 AM Retro Calibration 9 19 2012 10 49 08 AM Image Quality 20x Ch12 Test 9 19 2012 8 47 17 AM Autosampler Nest Calibration 9 19 2012 8 37 23 AM Image Quality 40x Ch 6 Test 9 19 2012 8 41 11 AM X Image Quality 40x Ch12 Test 9 19 2012 8 41 41 AM M hd Start All Tests Start All Calibrations 46 Utilities Utiity Last Run Time 405nm with LAF Laser Utility 8 12 2012 7 14 26 PM 488nm with LAF Laser Utiity en27a0t2 71514 PM 785nm Ch 6with LAF Laser Utity 8 12 2012 7 16 29 PM 785wmChi2wih LAF Laser Utiity 8 12 2012 7 18 16 PM Autofocus S Curve 20x Utility Autofocus S Curve 4 x Utiity 7 10 2012 11 00 01 AM Autofocus S Curve 60x Utiity 7 11 2012 7 12 37 AM Brightfield Calibration Utiity 6 27 2012 4 30 56 PM Compensation Utiity 8 13 2012 11 40 29 AM Cross Correlation 20x Utiity Cross Correlation 40x Utiity 7 31 2012 3 32 06 PM Cross Correlation 60x Utility EDF Excitation 20x Utiity EDF Excitation 40x Utility 8 8 2012 11 20 21 AM EDF Excitation 60x Utiity 8 8 2012 11 22 48 AM Focus Offset Beads 20x Utility Focus Offset Beads 60x Ltity 7 11 2012 6 45 48 AM Focus Offset Cells 20x Utiity Start All Calibrations
71. s are stained with more than one fluorochrome 75 Chapter 5 Software BH NW already running Imaging is paused Click Resume No objects in the current image view mode In the cell view area select the all pop ulation Wait until the script completes or if nec A scriptis running essary click Abort Script to prematurely stop the operation Open the Windows Task Manager by pressing Ctrl Alt Del Click the Applications tab If INSPIRE is Not Responding select the INSPIRE task and click End Now Restart the INSPIRE application by double clicking the icon on the desktop If the program restarts make sure the lasers and bright i4field lamp are turned on and then re establish the core stream If the appli cation does not start use the Windows Task Manager to end the INSPIRE task again Shut the instrument and com puter 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 before running sterilize On the keyboard press Ctrl Alt Delete INSPIRE fails to Splash screenis Jopen the task manager select INSPIRE launch not responding and press end task Wait 60 seconds and try restarting INSPIRE Loss of com Shut down the computer and power off munication the instrument Verify all computers are between the com Joff Power on the instrument and the puters and instru computer wait 5 mi
72. s by returning any sample and then sopping and running The two brightfield Frame Once E fluidics Load the default template and images are not of verify brightfield is in channel 1 and 9 the same cell at 800 counts of background Open ASSIST re run the frame offset cal ibration routine and verify it passes Call service and verify that the illu mination pathways are in proper align ment Run SpeedBeads by returning any sample and then sopping and running fluidics Load the default template and verify brightfield is in channel 1 and 9 at 800 counts of background Open ASSIST re run the cross correlation utility and verify it passes Illumination is grossly misaligned Cross correlation is incorrect Images appear pix elated or larger Image gallery zoom Use the magnifying glass to zoom is active out and restore the native image size In the magnification and EDF section choose an appropriate magnification for your cell type Use the magnifying glass to zoom out and restore the native image size To activate a channel for acquisition click on the channel column heading i e Ch2 and check the collected check box to save that channel Image gallery dis Click on the channel column heading play is set up incor i e Ch2 and set the display and chan nel color for the channel 82 Fluorescence imagery appears Fluorescence is too bright images have a contrasting Intensity Image display
73. sheath then go to the instrument empty drop down and run prime With the system powered down look for Fluidics Air buffer in the respond slug sheath syringe is gishly not correct Event rate slows Cells have settled in the lines Make sure the sample concentration is between 107 and 108 cells ml Lower con centration is low centrations can be used but this will decrease the cells second Cropped images will be eliminated from data acquisition and if enough of the images are cropped the event rate can appear lower than normal Normally this is due to air in the system Run the purge bubbles script from the instrument drop down menu See solutions for unstable fluidics Core is off center 72 Possible Causes Recommended Solutions Cross con tamination from Erroneous fluid level indicator Instrument will powers to maximum and decrease them to prevent pixel saturation For large diameter cells go to the advanced drop down select acquisition and check the box labeled keep clipped objects DNA dyes must be thoroughly flushed from the sample lines to prevent residual dye from labeling subsequent samples Load a sample of 1096 bleach followed by a PBS wash to remove all traces of the DNA dye in the instrument or run the sterilize script 30min Cells from the pre This suggests a minor clog Load a sam vious sample are ple of 10 bleach followed by a PBS appearing in current wash to remov
74. tion 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 56 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 E Brightfield Intensity Selection Test Parameters Num Settings Channel Df Interest Num samples E Total Range Channel vs Iterations 2 2 E m e 5 5 E 5 Z Idle Database Setting Saved Value Result Value ASSIST BFTestQualityMin 0 00 ASSIST BFTestQualityMax 20 00 ASSIST BFTestQualityCh1 8 00 ASSIST BFTestQualityCh2 3 00 ASSIST BFTestQualityCh3 7 00 ASSIST BFTestQualityCh4 3 00 ASSIST BFTestQualityCh5 3 00 Start The results and limits of the test are shown below of the list when the test is selected 57 Chapter 4 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 ac
75. tions and Tests Start All 45 camera 12 88 Centering adjustment 23 Cleanser 14 Compensation button 18 mode 33 Core Tracking 28 data files 32 Debris Squelching 35 Debubbler 14 DNA dye cleaning 33 EDF characteristics 37 turing on 31 using 37 Features Area 31 Aspect Ratio 31 Background Mean 32 Gradient RMS 32 89 Chapter 6 Intensity 32 Major Axis 32 Mean Pixel 32 Minor Axis 32 Object Number 32 Raw Centroid X 32 Raw Centroid Y 32 Raw Max Pixel 32 Raw Min Pixel 32 Time 32 Uncompensated Intensity 32 fluorochrome balancing 30 Focus adjustment 23 Graphs features 31 histogram 18 refresh 18 scatterplot 18 Image Display properties 17 Image Display tools 16 Image Gallery 16 pause button 21 tools 16 90 ImageStream shutting down 36 starting 28 INSPIRE User Interface 15 Instrument Control Panel 19 laser 12 Laser adjustments 22 31 Magnification changing 22 31 Mask display 16 Menus Advanced 26 Analysis 26 Compensation 26 File 24 Instrument 24 Layout 26 MultiMag 38 optimizing settings 13 91 Chapter 6 Optional settings 35 Options 37 MultiMag 38 password 28 Physical requirements 2 reagents 14 Run beads 45 Run Setup mode 21 Safety 2 Biological Safety 9 Electrical Safety 6 Laser Safety 7 Sample buffer compatability 30 load 21 order 30 Saturation color 17 sheath bottle 14 sheath fluid 14 Shutdown
76. tive under Focus and Centering Core Tracking is adjusted by pressing right or left arrows to center images 28 Daily Operations 9 The event rate should be 800 1000 events per second If not see Trou bleshooting Note Instrument calibrations may also be run individually by selecting a particular procedure under Calibrations or Tests Next to each calibration or test button is a green or red rectangle If the procedure fails it turns red If a procedure fails repeat it If it fails twice see Chapter 5 See System on page 69 or call your Amnis Field Service Representative For more information on the individual calibrations and tests referto theFigure ASSIST Calibrations on page 47 in chapter 4 10 When the calibrations and tests have passed the ASSIST status light will change to green Close the Calibrations window 29 Chapter 3 Data Acquisition After the ImageStream system is calibrated 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 Refer to the ImageStream Sample Prepar
77. ttings Squelching Debris Some samples have an abundance of small particulate debris These can be eliminated from collection by gating or by using Squelch to reduce the sensitivity of object detection As opposed to gating 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 4000 Squelch values range from 0 to 100 increasing the value decreases object detection sensitivity 1 Choose All in the image gallery 2 Observe the relative proportion of cell to debris images appearing in the imaging area and the event rate Total Sec under Acquisition Status 3 Onthe Advanced Setup Acquisition tab increase the Squelch value until the observed proportion of cells to debris increases in the imaging area 4 ObDservethe Total Sec event rate on the Setup tab under Acquisition Status If it is still greater than 500 repeat step 2 Setting ImageStreamX Speed and Sensitivity The optimal operating speed is set at the factory for each instrument and is approximately 60 mm sec This speed corresponds to the highest resolution setting shown below with a pixel size of 0 5 uim at 40 X magnification In order to collect images at higher speed the rows on the camera can be binned The center of the control corresponds to 2x binning and the right setting corresponds to 4x binning The sam
78. tween 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 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 48 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
79. ty EMC Part 3 2 Limits Limits for Harmonic and Emissions Equipment Input Current 216 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 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 61000 4 3 2008 edition ompatibility Part 4 Testing and Measurement Techniques Section 4 Electromagnetic C Electrical Fast Transient Burst Immunity Test EN 61000 4 4 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 Disturbances Induced by Radio Frequency Fields EN 61000 4 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 11 bem Short Interruptio
80. 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 absence of light its signal is 30 counts Dark Current Calibration Parameters Number of T T D D D 1 600 800 1000 1200 1400 1500 Camera Pixel Column Database Setting Saved Value Result Value ASSIST DarkCurrent3_Gen2_2 21 3977 2 ASSIST DarkCurrent Max 100 000 The Dark Current calibration commands the system to tum 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 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 fro
81. wn from these bottles into the sheath and flush syringe pumps The sheath pump controls 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 and alternating with the sheath pump also controls the core 14 Fluidics INSPIRE User Interface The user interface is divided into 3 areas the image gallery where channel images are displayed a work area where graphs of features are displayed and the controls section where the instrument is controlled The layout of the Image Gallery and Analysis area can be vertical or horizontal and changed under the Layout menu Status information is displayed along the bottom of the window Image Gallery Instrument controls pn bos n Swich Cove Mode completed at 40 AM on 6 6 2012 San sa GD A AA ompersaton Q Fous Fow Assist 15 Chapter 3 The Image Gallery Images are displayed in the image gallery during setup and acquisition Image Gallery Tools All 1267 obj s lil aa VI Region of Interest cic Minimum 46 Options Maximum 80 Show Channels Mean 59501984 Show Alignment Tools 11058415 Std Dev Corrected 11058415 Sid Dev Diffed 0 1125 125 Area Pixels 1512 Intensity x E Area Microns Ptr Line Rgn Buttons that allow interrogation of pixel information of a single point Ptr a line or a region Rgn of the imagery Pixel Information box Displays the s
82. wn menu See solutions for unstable fluidics Re run the focus adjuster and frame offset calibration in ASSIST and verify it passes Camera line rate is incorrect Excessive core stream variation due to air or clog in the fluidics Core stream is mov Allow the system to settle for 60 sec ing too fast for the fonds after loading a sample Collect camera data once imagery looks good In the Focus and Centering section Autofocus is not adjust focus and centering left or right tracking properly until the images are centered and in optimal focus Tum off bin mode by selecting fluidics and set the slider bar to low speed Data is binned high sensitivity Verify this change in the advanced drop down by selecting camera and verifying a 1x bin mode uncheck the EDF checkbox Lateral deviation of the core stream due to air or clog in the system Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics In the Focus and Centering section EE adjust focus and centering left or right tering is not tracking until the images are centered and in properly optimal focus Incorrect Mag In the magnification and EDF section Autofocus and cen 81 Chapter 5 Possible Causes Recommended Solutions Jnification choose a lower magnification Run SpeedBead
83. y 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 ES Flow Core Axial Stability Test Parameters Num Settings Channel Of Interest Num samples Iu Total Range Axial Stability 0 10471 0 08 4 0 06 0 04 0 02 0 00077178 7 n 7 0 10 20 30 40 50 60 70 80 Sample Number 1 1 1 1 Restoring Instrument Settings Database Setting Saved Value Result Value ASSIST ASSIST FlowSpeedCV 0 03 ASSIST FlowSpeedCVMax 0 15 ASSIST FlowSpeedCV_PercentO verMax 0 10 Percent Over Max The flow core axial stability test plots 100 flow speed sample intervals each of which consists of an average vel

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