Home

INSPIRE

1. Image Gallery Tools Cu ET EX L CU Image Display Tools e Ptr Line Rgn Buttons that allow interrogation of pixel information of a single point Ptr a line or a region Rgn of the imagery e Pixel Information box Displays the selected Pixel x y coordinates and its Intensity value e 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 e Intensity Profile Plot of horizontal pixel number vs Mean pixel intensity for the drawn region 17 Fluidics 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 hi 6 Boss ED L Icon Name Pescription a Opens the bivariate scatter plot tool TU farmer prawan ovaregon ona seatepot Tie Ey ric IT Compensation Opens the compensation wizard Be e E Chapter 3 The Instrument Control Panel The instrument control panel provides tools to control instrument operation data acquisition and status a am Sample Time Remaining 41 15 Acquisition gt es a All 8 obj s Filename 060712 X197 MII Fite noBE_1 1rif Custom Filename Text Se
2. Load the plate into the Autos ampler and press the Start button Status Idle Idle Idle Idle Idle Idle Idle Idle Idle Idle Idle Idle Idle Idle 11 Check or uncheck the boxes Return sample or Sterilize Note that these boxes may be checked or unchecked while the plate is running and the operation will apply after the current sample is finished 12 Select the wells to run they will appear in the list 13 Click Open Door to extend the plate nest 14 Place your plate on the nest with well A1 positioned at the upper left corner 15 Click Start to begin 16 The Status column will be updated for each well as it is run For each sample the instrument performs the following in sequence 1 Flush Lock and Load 2 Val idation flow speed CV focus brightfield intensity object rate 3 Data Acqui sition 4 Result success or error 17 During a run e You may stop the plate at any time by clicking the Stop button This does not initiate sterilize even if the Sterilize after running plate box is checked _ A5 Chapter 3 e Should the sheath tank or beads reservoir become empty or the waste tank full during a run an alert will be sent to the email entered in the well plate def inition Acquisition will pause until the user intervenes e fan 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 autosampl
3. a cece cece ceccceeececceseeeeseceenees 56 ASSIST Tests iii iii diese iii eue eicees nue 57 Excitation Laser Power Tests c cece cece cece e cece e cece eeeceeeeee 57 BF Intensity Selection Test 00000000000 rrr2a1220211n 58 BF Uniformity TESSU Es ns a a r easel a 59 Flow Core Axial Stability Test 60 Flow Core Lateral Stability Test 62 Flow Core Position Test 63 MOIRA AA 64 Image Quality Ensquared Energy Test 66 ASSIST Utilities eee 68 Autofocus S Curve Utility 22 68 Brightfield Calibration Utility 22 70 EDF Excitation Utility 2222222 11 FOCUS Pan UU sense ne nopisaana babe woud noes ae ns 13 PMT Focus LION nan sement ie nement 14 S Curve Peaks Utility 2 0 0 000000 c ccc cece cece ccc ccc ccc cccccecceeceeeeeeseeeeee 15 Troubleshooting zzz TI 5 6 paaa AN Aa Bak BASA Na a AA Uma AA AAO 7 D ON AR a dE CL 7 AS AA Tf Intensity 22222 idee 7 Chapter 1 Information and Safety This section covers safety information for operating the Amnis ImageStream multispectral imaging flow cytometer Anyone who operates the ImageStream should be familiar with this safety information Keep this information readily available for all users The safety information consists of the following areas e General Information and Safety e Explanation of Symbols e Electrical Safety e Laser Safety e Biological Safety Chapter 1 General Information and Safety The ImageStream
4. Template Error Notification File Email Customize Well Plate SelectbyColo EIR RM Select Defined Delete Selected Wells afaj ea ezel oojaa e CIE IE E O O O O O E E 7 a o e gt l El Gi El E required for complete well definition 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 Cell Count and Template File in order to be considered defined Other param eters can optionally be added to the definition in the next step 5 Choose the parameters you would like to use e Click Add Remove Well Parameters to choose the parameters you want to report for the wells 42 Optional upgrades Add or Remove Columns Standard Parameters Custom Parameters C Display Parameters Fluidics Parameters C Illumination Parameters Imaging Parameters Enter custom column name here Add Custom Column OK Cancel There are several categories of parameters that may be chosen as a group or individually See the list of parameter below Blue Well Parameters on page 46 Check or uncheck the desired parameters The user can also define cus tom parameters Expand the category to see the individual parameters To delete a custom parameter select it and use the delete key Click OK when
5. A8 JA08 TE Ble C Documents and Settings amms Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amnis com CD45F TNF Untx 60min B1 B01 8 ATE Bed C Documents and Settings amris Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF A 100pg 15min B2 B02 TE Bed C Documents and Settings amnis Desktop SX D ata 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF B 10pg 15min B3 B03 f Ped C Documents and Settings amms Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF c lpg 15min B4 BO4 Bed C Documents and Settings amris Desktop ISX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF A 100fg 15min 8 Highly recommended select Error notification Email from the list of Standard parameters and type in the user email address in the Apply to Selected box 9 When done click Save 10 Click Start to run the plate e A warning may be displayed if there are undefined or partially defined wells Select Yes to return to plate definition or No to continue INSPIRE Template File Not Specified Some wells have only been partially defined Wells that are not Fully defined will not be included when running plate definition Would you like to return to the definition screen to edit the plate _ A4 Optional upgrades The Auto S
6. Parameters Hum Settings 40 Channel Of Interest Hum samples 5 E Total Range 40 l l 2 3 4 5 E 7 a 3 10 PT 1 10 g TI o 1 E PMT Energy 4 2 0 Focus Position um PMT Energy 0 TI o 1 2 3 4 5 6 T pa g 10 Focus Position um The results and limits of the test are shown below the list when the test is selected 75 Troubleshooting 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 e Unstable fluidics Air or clog in system e Fluidics respond sluggishly e Event rate slows over time e Event rate is slower than expected e Cross contamination from previous samples e Erroneous fluid level indicator e Instrument will not pass ASSIST e Compensation wizard fails to complete Software e INSPIRE appears to freeze e INSPIRE Fails to launch e Plots fail to update or update slowly e Data file fails to collect Image e Noimages e maging and acquisition rate is erratic or appears frozen e Objects appear streaked e Objects are not centered in the channel e Objects are rotating in the core stream e Objects are out of focus or distorted e Objects are cropped e The two brightfield images are not of the same cell e Images appear pixelated or larger than normal e Objects appear larger or smaller than normal e Not all 12 channels are being disp
7. each channel Grating spacing and peak signal levels are reported to the interface The utility is not run as part of the routine ASSIST tests Es PMT Focus Utility Parameters Hum Settings 40 Channel Of Interest Hum samples 5 Total Range 40 PMT 1 10 a E PMT Energy 4 2 0 l l l l l l l l l l o 1 2 3 4 5 6 7 ai J 10 Focus Position um PMT Energy 0 TI o 1 2 3 4 5 6 T pa g 10 Focus Position um 74 S Curve Peaks Utility Measures the depth of modulation of the AFFS S curve The autofocus system provides an error signal that proportional to defocus in microns When the error is O the system is in the best focus position The error signal theoretically modulates between 0 9 and 0 9 with a linear error response centered around best focus As objects are moved continuously away from focus in either direction the S curve will peak with a negative or positive value at positions 7 5 microns from best focus After this point the error signal will begin to decrease and eventually drop off to O again The S curve peaks test pans through 15 microns of focus and measures the peak response in the negative and positive directions The peak responses are tested against limits to ensure that the focus system provides enough modulation depth to pull objects into focus The positive and negative peak values are stored in the ASSIST database Es PMT Focus Utility
8. is active Incorrect mag nification Image gallery zoom is active Channel is not acti vated Image gallery dis play is set up incor rectly Images have incor rect colors Frame offset is incor Troubleshooting Recommended Solutions Poo nification choose a lower magnification 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 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 Use the magnifying glass to zoom 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 Click on the channel column heading i e Ch2 and set the display and chan nel color for the channel 89 Chapter 5 Intensity Recomm
9. ASSIST Load Sheath Flush Load Beads Load Flush Springe Prim Purge Bubbles View Tank Lewels View Compensation Matrix Senace Scripts k 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 28 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 View Tank Levels Opens the fluid level window Service Scripts For field service personnel only Options INSPIRE Options Email Notification SMTP Host SMTP Port 23 Login Password Enable SSL e Analysis menu Access the Feature Population and Region Managers Func tionality is the same as for IDEAS Refer to the IDEAS user manual for more information Analyss Features Populations Regions Features Opens the Feature Manager Features can be renamed or new combined features can be created Populations Opens the Population
10. Backup and delete data to free up disk space Some samples have high con centrations and acquire faster than the Parent population has no qualifying events Data file fails to 83 No events qualify collect for the region Computer hard drive is full Data file collected rapidly display rate Check the destination folder and see if the raw data was col lected 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 rif or fcs file Go to the file drop down menu and Was created check Generate rif and or fcs file File directory was lost 84 Troubleshooting 85 Chapter 5 Image Symptom Possible Causes Recommended Solutions If the camera is Click Stop to stop the camera and already running then click Run Setup Imagingis paused Click Resume Displayed region is In the cell view area select the all pop incorrect ulation 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 Make sure the brightfield lamp is turned on and click Set Intensity Core stream is out Manually find the core stream In the side the objective s focus and centering section m
11. Calibration Parameters Num Settings 100 Channel Of Interest 6 Num samples 200 E Total Range 24 Power vs Intensity GB H Power mw Database Setting Saved Value Result Value ASSIST SideScatterLaserlntensityMin 6 400 00 ASSIST SideScatterLaserlntensityMax 8 000 00 AS515T Side5catterLaserlntensityT arget 7 200 00 ASSIST Side5catterCaliblntensity 6 989 29 ASSIST SideScalterC alibPower 1 92 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 pita Retro Calibration Parameters Hum Settings 30 Channel OF Interest i Hum samples 500 FE Total Range 1 50 EE E000 Total Intensity 3000 2233 5 T l l l l l l 200 B60 A40 B20 B00 its r60 Retro Position um Idle Database Setting Saved Value Result Value ASSIST EsxcPo
12. DI water for rinsing the instrument during shutdown Fluid is drawn from these bottles into the sheath and flush syringe pumps The sheath pump helps to control the speed of the core stream and the size of the core stream diameter The flush pump is used to clean and flush the system and alternating with the sheath pump also controls the core 20 Fluidics INSPIRE User Interface INSPIRE for the File Instrument A Ri Channa 1 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 ach Core Mode completed at 8 40 AM on 6 6 2012 Work Area R amp ip gt 3 a 71 Li amp ai to 53 EX penosa Analysis Layout Advanced Help NI il a Qs Sample Channel 2 Channel 3 Channel Channel KAG gt kd T Les Sample Time Remaining 2 Acquisitior 640 Elapsed Acqusit m Filename 1 i me lt 051 2 a i N Channels LS Illumination 405 1207m Brightheld 642 100 ni Set Intensity 255 100 2 m Fluidics Chapter 3 The Image Gallery Images are displayed in the image gallery during setup and acquisition
13. DNA dye from pre tamination from vious sample is labeling current 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 level indicator is correct Power down and power up the instru Sensor is broken ment if this does not fix the problem call Amnis service Erroneous fluid level indicator Instrument will not pass 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 ante 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 Go to the file drop down and select load default template Re run ASSIST The particles must be running gt 1000 SpeedBeads are 5s 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 80 Troubleshooting Possible Causes Recommended Solutions 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 Calibration and or run the test by clicking in the box next to test failure the tes
14. Stream is rated at BSL1 Do not load or flush samples containing infectious agents without first exposing the sample to inactivating conditions It is recommended that samples be fixed in 2 paraformaldehyde for at least 10 minutes before running the samples on the ImageStream The use containment and disposal of biologically hazardous materials are required to be in accordance with Personnel Protective Equipment Directive 93 95 E and are 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 Securite BiologiqueBiorisques L image Stream est valu a un niveau de s curit biologique L1 Ne pas acqu rir ou vider des chantillons contenant des agents infectieux sans les avoir inactives II est recommand que les chantillons soient fixes dans du paraformaldehyde 2 pendant au moins 10 minutes avant d acqu rir des chantillons avec lImageStreamxX 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 util
15. To use the Autosampler for unattended operation see Using the Autosampler Refer to the ImageStream Sample Preparation 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 e Experimental sample with the brightest stains to set the sensitivity for the run e Single color DNA dye control NO BF or SSC to ensure correct dye con centration e 10 bleach to wash out DNA dye followed by PBS e Single color fluorescence controls no DNA dye NO BF or SSC e The rest 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 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
16. aaao 00000A AAAA LLDD LD L aaan aaao 33 Sample order aaao aoaaa anaana anaoa oaoa 000a aaa A AALL L DDL D DDADL LLL LLL nnana 33 Loading and running the sample 2 2 2 a 33 Collecting and saving the data files 000000000000000000000000000000000000000n 35 Optional SSMS AA 37 Setting the Image Display Properties 2 2 22 c cece cece eee e eee 37 Squelching Debris 222 3 Setting ImageStreamX Speed and Sensitivity 2 22 22 eee 38 Daily Shutdown Procedure 2 000000000 0000000000000 ccc ccec ccc ecceccecceeeeeesees 39 Optional upgrades cc ccc ec cece eee eeeeceeeceeeeeeees 40 EE Oe D RE PA 40 To collect a data file using the EDF element 0 0 00 000 22 22 222222 40 Using MultiMag 111101100 02 0 ccc cence ccc n cence ccc ecceceecceceeeeeeeeeeeeseeseeees 41 Using the Autosampler 222 41 TODE HI AA 42 AS DOT AA PA 47 ASSIST Calibrations LL 49 Camera Synchronization Calibration 0 77701 0 02 2 Q X a 49 Spatial Offsets Calibration 2 50 Dark Current Calibration 2 0 0 0 0 22 c cece cece ccc ce cee ec cececceceececeececees 51 Brightfield Crosstalk Coefficient Calibration 0 00 00020 202 c eee e eee 53 Horizontal Laser Calibrations 00 0 00 00 ccc cece eee cece eee ce cece eceeeeeee 54 Contents Side Scatter Calibration 2 2 0 0 cece eee e cece eee ceecececeeseceeeeee 55 Retro Calibration Z
17. calibrations and tests 2 Optional Click Run Beads to begin running beads without starting calibrations or tests 3 Torun one calibration or test click on an individual calibration or test and click Run 4 To stop a calibration or test click Stop or Stop All if Start All was chosen A calibration or test will be flagged red if it fails If a calibration or test fails run that calibration or test individually and if it fails again call or email Amnis service Note Calibrations and tests do not run in order 40X Calibrations are completed before changing magnifications to run 20X and 60X calibrations 47 Chapter 4 Calibrations Calibration Camera Synchronization 20x Calibration Camera Synchronization 40x Calibration Camera Synchronization 60x Calibration Spatial Offsets 20x Calibration Spatial Offsets 40x Calibration Spatial Offsets 60x Calibration Dark Current Calibration Brightfield XTak Coefficient Calibration Core Stage Position Calibration 405mm Horizontal Laser Calibration 488nm Horizontal Laser Calibration 561nm Horizontal Laser Calibration 592nm Horizortal Laser Calibration 642nm Horizontal Laser Calibration 785nm Horizontal Laser Calibration Side Scatter 20x Calibration Side Scatter 40x Calibration Side Scatter 60x Calibration Last Run Time 6 14 2012 2 15 52 PM 6 14 2012 2 03 02 PM 6 14 2012 2 14 12 PM 6 14 2012 2 16 15 PM 6 14 2012 2 03 36 PM 6 14 2012 2 14 32 PM 6 14 2012 2 03 40 PM 6 14 201
18. ee Template File EnorMotiication Stain Treatment Comments Patientit Dose Time Include in filename yes no no no yes yes no yes yes yes Apply to selected DATE ic Documents and Settings amnis Desktop ISX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amnis com CDA45F TNF Al JA01 R 101 DATE Blue C Documents and Settings amnis Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF Fite ctrl 100pg 60min A2 A02 It Blue C Documents and Settings amrus Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist armris com CD45F TNF PE ctl 10pg 60min A3 20314 A T Blue C Documents and Settings ammis Desktop ISX D ata 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amnis com CD45F TNF AF647 cul Ipg 60min AA AD4 T Blue C Documents and Settings amnis Desktop ISX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amnis com CD45F TNF 100fg 60min A5 105 T Blue C Documents and Settings amrns Desktop ISX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amnis com CD45F TNF 10fg 60min A6 20614 T Blue C Documents and Settings ammis Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist amris com CD45F TNF DAPI lig 60min A7 AO7 IH TE Slee C Documents and Settings amnis Desktop SX Data 043010 X101 WB MONO NFkB TNF 15 5000 043010 WB CD45FITC ist arnris com CD45F TNF 10Dag 60min
19. 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 Cells are stained with more than one fluorochrome 82 Troubleshooting Software Possible Causes Recommended Solutions If the camera is already running NSPIRE appears Camera IS not run Click Run Setup to freeze ning Click Stop then Run Setup Imaging is paused Click Resume No objects in the current image view mode A script is running The INSPIRE appli cation has crashed INSPIRE fails to launch Splash screen is not responding Loss of com munication between the com puters and instru ment In the cell view area select the all pop ulation Wait until the script completes or if nec essary click Abort Script to prematurely stop the operation Open the Windows Task Manager by pressing lt Ctrl Alt Del gt 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 field 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 th
20. histogram 23 refresh 23 scatterplot 23 Image Display properties 37 Image Display tools 22 Image Gallery 22 pause button 25 tools 22 ImageStream shutting down 39 starting 31 INSPIRE User Interface 21 Instrument Control Panel 24 96 laser 18 Laser adjustments 26 34 Magnification changing 26 33 Mask display 22 Menus Advanced 30 Analysis 29 Compensation 30 File 28 Instrument 28 Layout 29 MultiMag 41 optimizing settings 19 Optional settings 37 Options 40 MultiMag 41 password 31 Physical requirements 8 97 Chapter 6 PMT settings 74 R reagents 20 Run beads 47 Run Setup mode 25 S Safety 8 Biological Safety 15 Electrical Safety 12 Laser Safety 13 Sample buffer compatability 33 load 25 order 33 Saturation color 37 sheath bottle 20 sheath fluid 20 Shutdown 39 button 27 SpeadBeads run 47 Speed control 26 38 SpeedBeads loading 31 98 Startup 31 button 27 Sterilizer 20 Taskbarinformation 27 Templates 33 Test BF Intensity Selection 58 BF Uniformity 59 Excitation Laser Power 57 Flow Core Axial Stability 60 Flow Core Lateral Stability 62 Flow Core Position 63 Focus Offset 64 Image Quality 66 Tests 57 user name 31 Utilities 68 Utility Autofocus S Curve 68 Brightfield Calibration 70 EDF Excitation 71 Focus Pan 73 PMT Focus 74 S Curve Peaks 75 99 Chapter 6 waste 11 15 bottle 20 fluid 20 Weight 8 100
21. 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 z Brightfield Intensity Selection Test EF 0 Eg Parameters Hum Settings Channel OF Interest Hum samples Total Range Channel vs Iterations W3 o a o T o E Idle Database Setting Saved Value Result Value ASSIST BETestClualityMin 0 00 LASSIST BFTesiQualityblax 20 00 ASSIST BFTestQualtyCht 8 00 ASSIST BFTestQualtyCh2 3 00 ASSIST BFTestQualtyCh3 7 00 LASSIST BFTesiQualityCh4 3 00 ASSIST BFTestQualtyChS 3 00 The results and limits of the test are shown below of the list when the test is selected 58 BF Uniformity Test Measures the static and temporal uniformity of illumination in all brightfield channels channels 1 through 6 1 12 if the Twelve Channel option is installed Non uniformities in illumination can affect segmentation and the accuracy of photometric absorbance measurements made in the brightfield channel Non uniformities can be caused by misaligned illumination and collection path elements degradation of pixel responsiveness and electronic noise The brightfield uniformity test measures the response from each pixel column the illumination and collection systems are providing a uniform photometric response Es Brightfield Uniformity Test Parameters Num Settings Channel Of Interest Num sample
22. position in pixels is printed in the test window bas Flow Core Lateral Stability Test History 9 25 2009 9 13 05 4M Parameters Num Settings Num samples Total Range Std Dev Bead Position Histogram D 5 2 a o un l 0 10 x Centroid um Database Setting Saved Value Result Value ASSIST ASSIST LateralPositionStdDeyT estYalue 5 00 BO ASSIST LateralPositionStdDevT estMax 15 00 Camera CameraPixel5ize 0 50 Contaminated sheath obstructions air or improper pump function may broaden the core which can reduce focus consistency and increase variation in intensity measurements This flow core lateral stability ensures the core is operating as designed with minimal variation Failure to pass this test is indicative of at least one of the issues listed above The result and the limits for the calibration are shown below the list when the calibration is selected 62 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 determi
23. 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 90 Troubleshooting Symptom Possible Causes Recommended Solutions RE PE TT TR fa the brightfield intensity to 800 counts by pressing Set Intensity Load sheath then go to the instrument is empty drop down and run prime 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 The best instrument setup maximizes the dynamic range of fluorescence sig Instrument sen nal while at the same time avoiding sitivity is not opti 8 8 image pixel saturation which cannot mized be compensated In general decreas ing the laser powers until no pixels sat urate One channel sat urates while the others do not Scatter is too dim 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 Probing protocol requires better stain balance ever if too much dye is in solution it Excessive flu can cause the core stream to flu orescent dye is left oresce It s important to balance the in the sam
24. verify the appropriateness of the settings for the specific experimental run 3 Choose the objective under Magpnification option 233 Chapter 3 Q Magnification Magnihicaton 20X a0 60x 4 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 a Illumination 405 ap 120 005 mw Brightfield a 200002 mw f 7 642 10 00 mw Set Intensity 1000 E mw 5 Select Brightfield channels Default is Ch1 for a 6 channels system and Ch1 9 for a12 channel system Click Set Intensity 6 Select EDF collection if desired See Using EDF for details 7 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 e Ar
25. 0 5 0 4 D Lui o o io T Ww 0 3 0 2 0 14373 1 0 5 1 1 5 2 Focus Position um Idle Database Setting Saved Value Result Value ASSI5T CollectionlmageQualityFocusT est_LimitMin 0 55 AS515T Collectionl mageQualityFocusT est LimitMax 1 00 AS5515T Collectionl mageQualityFocusT est PercentUsed 0 02 ASSIST Collection mageQ ualityFocusT est Value 0 68 OM ASSIST CollectionlmageQualityFocusT est_VertRatio 0 88 DE 8SSIST CollectionmageQualityFocusT est HorizRatio 0 83 08 282820 v AS515T CollectionImageQualityFocusT est YertAngleT op 1 783 54 The test also reports regional scores which are not tested against limits The scores include the energy ratios for line profiles in the horizontal and vertical axes displayed at the bottom of the regional score grid and summed energy values for the horizontal vertical and diagonal directions radiating outward from the center of the image The summed energy values are displayed in a 3 x 3 array The value in the center of the array is the ensquared energy ratio for the single pixel in the center of the image If the MultiMag option is installed and ImageQuality Ensquared energy test will be performed for each magnification 67 Chapter 4 ASSIST Utilities ASSIST Utilities are a special set of procedures that are used to enable the collection of data Unlike ASSIST Calibrations or Tests no parameters are set for later use or tested against predet
26. 2 2 04 02 PM 6 14 2012 2 04 09 PM 6 14 2012 2 04 39 PM 6 14 2012 2 05 09 PM 6 14 2012 2 05 34 PM 6 14 2012 2 06 22 PM 6 14 2012 2 06 49 PM 6 14 2012 2 07 16 PM 6 14 2012 2 16 32 PM 6 14 2012 2 07 40 PM 6 14 2012 2 14 45 PM Start All Calibrations Tests Test 405nm Laser Power Test 488nm Laser Power Test 561nm Laser Power Test 592nm Laser Power Test 642nm Laser Power Test 785nm Laser Power Test Brightfield Alignment Test Brightfield Uniformity Test Camera Noise Test Flow Core Axial Stability Test Flow Core Lateral Stability Test Flow Core Position Test Focus Offset Beads Test Focus Percentage Test Focus Unfomiy Test Image Quality 20x Test image Quality 40x Test Image Quality 60x Test 48 Last Run Time 6 14 2012 2 09 19 PM 6 14 2012 2 09 29 PM 6 14 2012 2 09 38 PM 6 14 2012 2 10 10 PM 6 14 2012 2 10 15 PM 6 14 2012 2 24 03 PM 6 14 2012 2 10 54 PM 6 14 2012 2 11 35 PM 6 14 2012 2 11 39 PM 6 14 2012 2 11 54 PM 6 14 2012 2 12 04 PM 6 14 2012 2 12 10 PM 6 12 2012 10 03 36 AM 6 12 2012 10 03 47 AM 6 14 2012 2 13 14 PM 6 14 2012 2 16 59 PM 6 14 2012 2 13 29 PM 6 14 2012 2 15 02 PM Start All Tests n History Utility 405nm with LAF Laser Util y 488nm with LAF Laser Utilty 785nm with LAF Laser Utilty Autofocus S Curve 20x Utiity Autofocus S Curve 40x Utility Autofocus S Curve 60x Utility Brightfield Calibration Utiity Compensation Utility Cross Correlation 20x Utility Cross Correlation
27. 40x Utility Cross Correlation 60x Utility EDF Excitation 20x Utility EDF Excitation 40x Utility EDF Excitation 60x Utility Focus Offset Beads 20x Utility Focus Offset Beads 60x Utility Focus Offset Cells 20x Utility Focus Offset Cells 40x Utility Last Run Time 6 11 2012 6 21 24 PM 6 11 2012 6 33 01 PM 6 11 2012 6 30 30 PM 5 17 2012 11 22 45 AM 5 17 2012 1 40 55 PM 6 11 2012 4 00 20 PM 5 17 2012 3 54 21 PM 6 14 2012 1 55 48 PM 6 14 2012 1 28 44 PM 6 14 2012 1 35 12 PM 6 13 2012 5 20 31 PM 6 13 2012 6 09 55 PM 6 5 2012 10 15 04 AM 6 4 2012 5 10 12 PM 6 14 2012 1 54 16 PM 6 11 2012 5 42 22 PM Start All Calibrations and Tests Stop All Run Beads 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 bas Camera Synchronization 40x Calibration Parameters Num Settings Num samples Total Range Camera Sync E D o 2 o 2 5 wo 0 4 0 31463 4 36 40 41 Camera Sync Idle Database Setting Saved Value Result Value ASSIST CameraSynecStart 38 80
28. AS515T CameraMagMin 36 00 ASSIST CameraMagMax 42 00 ASSIST CarmeraMag 38 82 As shown in the figure above the camera synch calibration measures SpeedBead ellipticity at numerous discrete camera synch settings and plots the camera synch setting horizontal axis versus the ellipticity vertical axis It then generates the best fit curve for a 4th order polynomial through the data and determines the horizontal location camera synch of the peak of the curve The peak occurs where the SpeedBeads appear round This setting is then stored and used for all subsequent image acquisitions The result and the limits for the calibration are shown below the list when the calibration is selected Please note that Camera Synchronization Calibrations will be done for each magnification present in the system 49 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 ImageStream must maintain sub pixel spatial registry between channels ES Spatial Offsets Calibration Saks Parameters Num Settings Channel OF Interest Num samples Continuous Mode Total Range aaa maaaninag uang I
29. B GBM Level indicator for pumps i Green indicates compensation is being Compensation applied Yellow calibrations and tests not run Red one or more calibrations or tests ASSIST failed Green all calibrations and tests have passed 27 Chapter 3 Menu Bar The menu bar is located in the upper left portion of the INSPIRE screen It consists of these four menus e File menu Load and save instrument setup templates A template contains instrument settings that can be predefined and loaded to simplify the instru ment setup process File 9 New Template c3 Load Template Gd Save Template Load Default Template Gererate PIF file Gererate 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 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 e Instrument menu Run the ImageStream camera and instrument specific fluidic scripts automated fluidic routines Instrument Calibrste with
30. Ch2Kernel Default 000000 To perform this calibration 1 CD Verify that the instrument has been initialized ASSIST has been run and the instrument has passed the set of calibrations and tests run with Start All Stop the fluidics by clicking stop on the Setup page Deselect the Ignore All Enabled option in the cell classifier if necessary In the file menu choose Open template and select the template named EDF Calibration Template ist Check the following critical settings Collection Filter EDF BF OFF Ex laser 100 mW Ch1 stage setting 256 Ch2 6 512 Percent Beads 10 Diameter 3 microns Cell classification f Chapter 4 3 9 parameters set channels 1 and 3 to area min 25 max 500 Peak Intensity Max z 1000 Click Flush Lock Load script to load 50 ul of EDF beads into the sample line To view images click Run Setup and choose the Cells view Imagery in chan nels 1 and 3 will display the characteristic L shaped spread of the light from the EDF beads as shown Note if single beads as shown are not being characterized in the cell classifier call Amnis support Wait until the flow speed CV is consistently less than 0 2 Yo The events per sec ond should be between 50 200 second Select the EDF Calibration in the ASSIST tab and click Start Calibration Click Start in the Calibration window 10 When the calibration has finished the kernels should look similar to the images shown in
31. INSPIRE ImageStreamX System Software User s Manual Version Mark II August 2012 For updates log in at www amnis com Chapter 1 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 WWW amnis com The Amnis ImageStream cell analysis system is for research use only and not for use in diagnostic procedures WWW amnis co
32. ST DarkCurrentM ax 100 00 Minimum aa Lg Maximum The Dark Current calibration commands the system to turn off the excitation laser and brightfield illumination The system then measures the mean signal value of each camera column from 1000 rows of data per column The difference between this value and 30 counts is stored for subsequent correction When the camera is operated at different stage settings 32 64 128 256 stages the dark current characteristics of a column of pixels can change Therefore values for all stage settings are stored total of 3072 values INSPIRE automatically appends the calibration values appropriate for the stage settings used during acquisition to the rif file The values reported on the ASSIST tab indicate the maximum variation detected from all test conditions The result and the limits for the calibration are shown below the list when the calibration is selected If the 12 channel options is 51 Chapter 4 installed the Dark Current calibration will be simultaneously performed for both cameras 59 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 value
33. Uae Part 1 General Requirements EN 61010 1 2008 2 edition AUTHORIZED REPRESENTATVE Izasa SA Aragon 90 08015 Barcelona Spam VATS ESA28114742 Contact Jose Maria Alonso 34 916 630 550 TEST FACILITY F Squared Laboratones 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 Ku pail sos Ao Authorized by Date 23 August 2012 Name Richard Esposito Title Quality Assurance Officer LULU LE LI LT IE LE LT LE LI LT LE LILI LELIIZIELI LEE LCELTIIE LILI IT LIN ETIT LT LIIELE LIETIE S LT3TDPEPDPLELDLERLErRLCT SLrp LS LS LrTSr Sb S rfC CSC3 E L SL ST tb LL 3T tT eii 3S ze 3 eee iurng 10 Explanation of Symbols Explanation of Symbols Table 1 Risk of exposure to trans Waste tank missible biological dis Power supply cover D Power supply Protective earth ground Visible and or invisible Laser Radiation Risk of exposure to haz Awol td Ep sie Inside surface of hood P ardous laser radiation Interior side panels near release mechanisms and next to hood latch release Risk of exposure to haz ardous laser radiation No laser radiation is On the back of the instru accessible to the user dur ment ing normal instrument operation 11 Chapter 1 Electrical Safety Equipment ratings The ImageStrea
34. ampler 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 Es Auto Sampler Unattended Operation EE Open Door hi Start Return sample after running each well Flate Definition IT esting 030910 101 5F EdiThisPlate Edit This Plate This Plate og n O we eje e e e eo o elalolulel gt o oN 6 66 e o olc eee oj 6 66 60 0 Acquisition well Time a A2 A3 Ad A5 AB A7 AB Ag B1 B2 B3 B4 B5 PEPPE File Hame 6 28 2010 A0 6 29 2010 402 6 29 2010 403 6 28 2010 BO eluie eluIe J J d M agnification rif ghdagnifcabon rif 6 28 2010 403 6_28 2010 404 _ 6_28 2010 A05 6 28 2010 ADE 6_28 2010 407 6_28 2010 408 M agnification rif M agnification rif Magnification gt rif Magnification gt rif M agnification rif M agnification rit Magnification rif Magnification rif 6 28 2010 B02 6 28 2010 B03 6 28 2010_B04_ 6 28 2010 B05 M agnification rif M agnification rif Magnification gt rif M agnification rif 6 666666 6 666666 sblabladeas M Sterilize after running plate Well Definition Select the defined wells that you wish to run
35. appropriate sheath solution 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 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 SpeedBeads fail to run 78 Troubleshooting Symptom Possible Causes Recommended Solutions The sheath syringe should contain 2 4 mi of air to buffer the movement of the sheath syringe Is not correct 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 ad hs in the sysjinstrument drop down menu See solu tions for unstable fluidics 1 Sarpi Syng EAS Load a fresh sample empty Sheath sy
36. cus error scores from 100 SpeedBeads The stage then moves one micron toward focus and collects focus error scores again The process is repeated until the stage reaches a position 5 microns beyond best focus The data are then fit to a 3rd order polynomial and the slope of the curve at the inflection point is determined This value determines the signal error corresponding to a given focus error through the typical operating region of the autofocus system It is used to determine each move the focus motor makes to correct focus errors The result and the limits for the calibration are shown below the list when the calibration is selected If the Multimag option is installed the instrument automatically multiplies the S curve slope by the appropriate factors to compensate for the changes in slope that result from the additional 60X and 20X magnification 69 Chapter 4 Brightfield Calibration Utility The Brightfield Calibration Utility measures and stores the light output response as a function of input current for each LED used in the Brightfield illumination system In this calibration the optical system is commanded to place itself in a specified configuration 40X mag highest contrast aperture setting 6 0mm s flow speed 256 level staging etc The input current is varied over a predefined range and the light output is measured at the camera The resulting data are fit to a linear equation which are then used by the system rapidly s
37. d 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 top 2 of all imagery is computed and indicated as a dark blue data point on the plot This result is tested against predetermined limits and reported on the Collection Image Quality test tab and in the popup window This value is stored in the ASSIST database A highly magnified composite image of the top 2 of all images is also generated and displayed on the popup window Each small square of light is an individual pixel approximately 0 5 microns on a side in object space This image generally shows a small amount of flair on the right hand side This is due to light scatter from the far side of the SpeedBead which is approximately 1um in diameter 66 TE Image Quality 40x Test Parameters Num Settings Bo Channel OF Interest B Num samples foo o pa Total Range 40 CE Image Quality Focus Position vs Ensquared Energy 0 82741 0 7 0 6
38. d select the pop ulation To add another population click the box 9 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 Acquire the data Acquisition Pilo ti a All 1593 oby s Filename 060211 F5101 PKH26 Comp Control a Imaging should be running if not Click gt to start imaging b Click to collect a data file 12 The data file s are automatically saved in the selected folder once the desired number of objects are collected To prematurely stop acquisition click The system prompts you to either dis card the acquired data or to save the collected data in afile The acquisition can be paused and resumed by clicking _ 13 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 en
39. dle Database Setting Saved Value Result Value ASSIST Dffsets Gen 0 08 0 120 ASSIST YOffsets Gen 0 03 0 00 0 ASSIST SpatiallftsetsM in 0 95 A55157 5palalUlisetsMax 0 35 ASSIST SpatialOffsets2ndhefMin 5 00 ASSIST SoatalOfsets2ndhefhiax 3 00 Chi amp Olfset The SpatialOffsets calibration commands the brightfield system to illuminate all 6 channels simultaneously and collects imagery from 1000 SpeedBead objects in each of the six channels 6000 images total It then performs a two axis autocorrelation between the imagery from channels 1 5 with the imagery from channel 6 Autocorrelation is an accurate algorithmic technique that identifies the point at which two images exhibit the highest degree of overlap The autocorrelation results in a vertical and horizontal coordinate for each image correlation These values are then processed to determine the mean coordinates to bring each channel into spatial registry with channel 6 and therefore with each other 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
40. done e Toinclude a parameter in the file name click in the box below the column heading make sure it says yes e Columns can be re ordered by click drag e Click OK when finished adding or removing parameters 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 43 Chapter 3 bas Well Plate Definition Ik Start Plate Definition Name a M gt Customize Well Plate att in Delete Selected Wells Select by Color Select Defined 0G O 66 6660609 66 600600 0 66 6606000 0 66 660600 0 666060006 66 660600 0 6660000 66 60606060 Oe 66 660660002 o ua Sk o O Ji b j mmk GO OO G6 O G6 Pa 6G GG DES ES PAS ba KAKA BAGAL 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 For example if you want to collect 1000 events for the selected wells type 1000 in the Apply to selected box below the Cell Count heading If you want to only apply this to a sin gle well type this value in the Cell Count box corresponding to that well Below is an example of a Well Plate Definition using several parameters and showing only defined wells Filename Wel Date Color Output File Path MAGA
41. e instrument as described Ifa 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 open the task manager select INSPIRE and press end task Wait 60 seconds and try restarting INSPIRE Shut down the computer and power off the instrument Verify all computers are off Power on the instrument and the computer wait 5 min and try launching INSPIRE 83 Chapter 5 Symptom Possible Causes Recommended Solutions Plots fail to Computer update or update resources are Close all third party software slowly 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 1x107 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
42. ea The number of pixels in an image reported in square microns e Max Gradient Intensity The value of the largest slope spanning three pixels in an image This feature measures image contrast or focus quality e Intensity The integrated intensity of the entire object image the sum of all pixel intensities in an image background subtracted e Mean Pixel The average pixel intensity in an image background subtracted e Raw Max Pixel The intensity value of the brightest pixel in an image no back ground subtraction e Raw Min Pixel The intensity value of the dimmest pixel in an image no back ground subtraction e Saturation Count The number of pixels in an image that have an intensity value of 4095 e Saturation Percent The percentage of pixels in an image that have an inten sity value of 4095 34 Data Acquisition Collecting and saving the data files Once the sample is running and the ImageStream is properly set up you are ready to acquire the data as a raw image file rif 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 C File Acquisition Custom Filename Text Seq 060712 X197 MII Fte 4 amp Collect 1000 alot M F Channels 8 Enter the number of cells you want to acquire after Collect an
43. em for use the following day Optimizing instrument setup for sample runs is also described here in detail Fluidics INSPIRE User Interface Daily Operations Data Acquisition Daily Shutdown Procedure Optional upgrades 19 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 Source Catalog Cleanser Couitercienze Eka 8546929 Coulter Debubbler 70 Isopropanoli VWR 42101 0 ee VWR JT9416 1 chlorite pes nvvogen 14190 provided for information only other sources of the same reagent may be used Waste Fluid The waste bottle holds all of the fluids that have been run through the ImageStream and can hold up to 1600 ml Add 160 ml of bleach to the empty waste tank It is recommended that the waste bottle contain 10 bleach when full and that the waste line remain immersed in the liquid at all times to prevent changes in the flow rate Sheath Fluid Two bottles are provided one labeled Sheath to be filled with phosphate buffered saline PBS with no surfactants for running samples and one labeled Rinse to be filled with de ionized
44. ended Solutions Possible Causes Fluorescence imagery appears Image display set 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 Fluorescence is too bright images have a contrasting color or appear flat Image display set tings are set too high Instrument sen sitivity is set too high tings are set too low 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 Sample did not Look at the sample with a fluorescent label well microscope Turn 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
45. er moves on to the next well e If the same error occurs on three consecutive wells the autosampler aborts the plate and sterilizes the instrument if the Sterilize after running plate box is checked 18 A report will be saved to the folder designated in the Output File Path of the plate definition at the end of the run either when it was stopped manually or completed the entire plate Well Parameters Standard Parameters M Late Mel Color Output File Path Max Acquisition Time min M Cell Count Template File 1 Error Notification Email C Comments O Dose O Time Custom Parameters LI Patient L Cell C Treatment 0 Stain _ Display Parameters C Ch01 0 cho2 Cho3 DI Chos CJ chos Cy chaos Cy chor C Choe C chos C Ch10 C Chi C Chi 46 Fluidics Parameters Percent Beads C Core Diameter OC Core Velocity J Illumination Parameters 0 EF Intensity C BF Channel C 405 55C Blocker C 405 Power LL 488 Power 7 561 SSC Blocker L 561 Power J 592 SSC Blocker C 592 Power C 660 SSC Blocker C 660 Power CL 785 Power C 830 Power Imaging Parameters DAperture C Auto Bead Detection J Binning C Cross Camera Reference Channel CJ INSPIRE BF Gains Comected LI Maanification C Save Bead File Q Save Debris File C Squetch Chapter 4 Instrument Calibrations and Tests ASSIST Tab ASSIST Automated Suite of Systemwide Ima
46. ermined limits The utilities are a simply a set of repeatedly used procedures that facilitate the collection of data for other alignments and tests The Utilities are not run as part of the automated Start All ASSIST commands Autofocus S Curve Utility The Autofocus S Curve is the response of the Autofocus system to changes in focus There is a broad region of this curve in which the focus error varies linearly with the amount of defocus This calibration measures and stores the slope of the linear region of this curve focus error score microns The Autofocus system in the ImageStream is capable of detecting focus variations as small as 50 nm To maintain diffraction limited performance the Autofocus system measures and corrects any deviations in focus When the Autofocus system detects a focus error it commands a stage to move the objective lens to correct the error The actual move distance is the quotient of the autofocus signal error and the slope of the response curve pata Autofocus 5 Curve Calibration Parameters Num Settings Channel Of Interest Num samples Total Range Aubofocus 5 Curve E Tai Ta un KG O Pai LL l l l l l l l l 4 2 o 2 4 6 ai J Focus Position um Idle Database Setting Saved Value Result Value ASSIST SCurveSlopeMin 0 12 ASSIST SCurveSlopeh as 0 28 68 The Autofocus calibration moves the stage approximately 5 microns out of ideal focus and collects fo
47. et the illumination in a given channel for given configuration when requested Brightfield Calibration Utility eo Brightfield Calibration Utility History AAAH Parameters Num Settings La Channel Of Interest eo Num samples D a Total Range 00 Database Setting Saved Value Result Value 70 EDF Excitation Utility Extended depth of field EDF is a novel technique used in a variety of applications including FISH spot counting where having the entire cell in optimal focus is critical to obtaining accurate results There are two steps to utilizing the 16um EDF first images must be acquired with the EDF element in place and second the data must be deconvolved using the EDF kernel prior to analysis This calibration of the EDF element generates and saves the kernels used by IDEAS to deconvolve the imagery Calibration of the element is done on installation and should be repeated by Amnis service when any optical changes are made bas EDF Excitation Utility Parameters Hum Settings a Channel Of Interest gt a Hum samples 250 R Total Range M400 Sampling Progress D Ha E iza tu ra m un l 6 Focus Position um Database Setting Saved Value Result Value ASSIST EDFCalib Value 0 00 ASSIST EDFCalib_Kermel idth 32 ASSIST EDFCalib_KernelHeight 32 ASSIST EDFCalib_ExKemnelOffeet 10 ASSIST EDFCalib_ExKernel Offset 22 ASSIST EDFCalib_E Chi kermel Default 000000 ASSIST EDFCalb_Ex
48. exci tation bandwidth 405nm laser excitation currently set to OFF and O mW of power 488nm laser excitation currently set to ON at 60 mW of power a 1500 El mw 642nm laser excitation currently set to ON at 150 mW of power Hao 5 mu Set Intensity 705 ja 19 00 ES miv 785nm laser excitation currently set to OFF at 5 72 mW of power This laser is for side scatter only Brightfield 0 Brightfield illumination is shown as ON in land9 channels 1 and 9 Sets the Intensity of the brightfield to 800 counts Q Magnification Magnification Select the magnification Note this is optional equipment 40X 60X Fluidics Lo Spee 7 Adjust the speed and sensitivity for the run Speed and Sensitivity are inversely related Speed Sensitivity Med speed is 2X binned Hi speed is 4X binned 26 Fluidics Focus and Centering Centering Focus and Centering can be adjusted da o 00 using the right and left arrows PRE Runs the shutdown script and sterilizes the system Bottom task bar Our om SE gt GE mention fox fiw asas Status buttons are displayed at the bottom of the INSPIRE window Switch Core Mode completed at 3 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 shan SN G
49. geStream Tests is a suite of calibrations and tests for critical subsystems operating within the ImageStream ASSIST performs specific calibrations and tests measuring evaluating and storing thousands of values to ensure all subsystems are operating within normal limits ASSIST permanently logs results for all tests and flags any parameters that are beyond specified limits It is run daily using SpeedBeads to ensure optimal performance of the ImageStream A calibration is a sequence of operations designed to measure and set internal parameters that are used to operate a subsystem Calibrations are used to optimize performance of a subsystem or place it in predefined state After a calibration is performed it is tested to determine whether the calibration values are within a prescribed range A test is a sequence of operations designed to measure the performance of a specific subsystem The calibration and test values and acceptable ranges are listed on the ASSIST display tab A failed calibration or test is flagged with a red box The history of any calibration or test can be viewed by clicking on the box to the right of the specific item Utilities are calibrations used by service technicians Run the Brightfield Calibration Utility if the Brightfield Intensity Selection Test fails Run ASSIST daily to optimize the performance of the ImageStream TorunASSIST calibrations and tests 1 Click Start All Calibrations and Teststo run all standard
50. here are blurred events due to streak ing these can be removed from the analysis using a focus gate 40 Optional upgrades e EDF images exhibit increased texture due to higher resolution Brightfield imagery is not as crisp in EDF mode as in standard mode e 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 Prelsize epthoffield NA JO bom sum um pig 20x fi20um hum Bum p5 sox Houmbssum sum po 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 nest for loading of samples in a 96 well plate format Prior to running the plate a plate definition is created that assigns instrument settings to the wells na
51. if the 12 channel option is present this calibration will illuminate and calibrate all 12 channels 50 Dark Current Calibration Measures and stores 3072 offset values corresponding to pixel columns in the TDI camera Every pixel in a CCD detector is an individual sensor with its own sensitivity characteristics In the absence of any light each pixel emits a signal known as dark current Although the statistical variation of any given pixel over time is less than one count the mean dark current signal generated by any pixel may vary as much as several counts from a different pixel in the array When the ImageStream is measuring very dim signals even one count difference between pixels can be critical Therefore a Dark Current calibration factor is stored for each pixel column This factor is added to or subtracted from each pixel in the rif file during cif creation to normalize detector variation In the cif each pixel is calibrated so that in the absence of light its signal is 30 counts Dark Current Calibration Parameters Num Settings Num samples Total Range Average Intensity per Camera Column T z LE Fa Ka D l l l 200 300 400 500 600 700 800 Camera Pixel Column Idle Database Setting Saved Value Result Value ASSIST DarkCurrentO_Gen2_2 22 121093 ASSIST DarkCurrentl_Gen2_2 22 683593 ASSIST DarkCurrent2_Gen2_2 22882812 ASSIST DarkCurrent3_Gen2_2 24589843 EE ASSI
52. ilter dropdown menu Adjust cell classification settings to accomodate using EDF The calibration kernels saved during the last EDF calibration will be appended to the file and the file name will be appended with EDF Cell images as they appear on the instrument with left pair or without right pair EDF element in place Raw Max Pixel values indicated in upper left EDF image of a small bead showing characteristic L shaped pattern General characteristics of using EDF e 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 e During acquisition and before deconvolution images will appear blurred into characteristic L shaped patterns and raw max pixel values will be lower with EDF than with standard mode collection e Compensation controls for EDF data can be collected with or without the EDF element in place e When analyzing data in IDEAS after the deconvolution process there will be more light per pixel than in non deconvolved imagery Therefore raw max pixel values may exceed 1023 for the IS 100 instrument or 4095 for the ISX As long as the images did not saturate the camera during acquisition these pixel values are valid e Object Morphology and System Masks will be smaller in EDF mode e Focus gating is not required However if t
53. imaging flow cytometer is manufactured by Amnis Corporation andhas 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 80 for temperatures up to 31 C with the maximum relative humidity decreasing linearly to 50 at 40 C The main s supply may not fluctuate more 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 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 Us
54. ing gloves clean the sample portal and sample elevator with a 10 bleach solution Dispose of waste using proper precautions and in accordance with local regulations Preventative maintenance The ImageStream contains no serviceable parts Only Amnis trained technicians are allowed to align the laser beams or otherwise repair or maintain the instrument The instrument fluidic system is automatically sterilized after each day s use This reduces the occurrence of clogging Tubing and valves are replaced by Amnis service 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 fora Class A digital 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 no
55. isateur Respectez la r glementation en vigueur pour le traitement et l limination des d chets dans des r servoirs pr vus cet effet Pr venir l accumulation des d chets en vidant le r servoir lorsque l indicateur indique qu il est plein St riliser les instruments de routine apr s chaque journ e d utilisation Notez que cette proc dure ne garantit pas la st rilit vis 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 15 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 ImageStream system incl
56. ject detection As opposed to gating debris away from cells squelching debris can AT Chapter 3 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 On the Advanced Setup Acquisition tab increase the Squelch value until the observed proportion of cells to debris increases in the imaging area 4 Observe the 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 um 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 same amount of total intensity is collected Image resolution and sensitivity are inversely related to one another Lo speed Hi Hi Sensitivity Lo 38 Daily Shutdown Procedu
57. k 060712 X197 MII Fte aS 45 1200 mW Brightfield GED o 2 nv Gta 10 00 mw Setintensity 1000 H mw _ 24 Fluidics In the Sample section you can load a sam j vo Return ple or return a sample ae Sample time remaining is displayed when 4 Sample Time Remaining 5 34 a sample IS running Acquisition In the Acquisition section you can run in gt O ul LI setup mode begin acquisition pause or HU HU stop acquisition The Filename and the population and the rate of the population Filename 060211 F5101 PKH26 Comp Control being collected is displayed All 1593 oby s Run Setup Mode Imaging 7 FileAcquisition Acquisition In the File Acquisition section you can type in a custom filename set the Custom Filename Text 060712 X197 MII Fie sequence choose the data file folder type the number of events and choose the population to collect Collect 1000 Channels Filename Text Type the filename Choose the beginning sequence number a O Navigate to the folder to save the data Enter the number of events to collect a rr Can Add a second population to collect 25 Chapter 3 Illumination In the Illumination section you can turn 405 00 my lel i 3 z a 3 i Brightfield laser and brightfield illumination on or off jao 600 H mw kanda and set intensities All lasers have var iable power and are defined by their
58. layed e Images have incorrect colors Intensity e Fluorescence imagery appears too dim e Fluorescence is too bright images have a contrasting color or appear flat e One channel saturates while the others do not e Scatter is too dim or bright or changes over time e Large variation in brightfield intensity levels Brightfield intensity level sets incorrectly 77 Chapter 5 System Possible Causes mended 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 Clog in fluid lines 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 Sample is too con bles Dilute the sample to 1x10 7 Air bubbles in fluid lines centrated cells ml and strain the cells through a 7Qum nylon mesh Run the purge bub bles script Verify the sheath is dPBS De gas the sheath as appropriate Third party sheath buffers cannot be used In
59. m Contents Contents Patents and Trademarks 0000 anaana 1 aa01aan11nn 2 DISCO aan Bina aa 2 Technical Assistance 2 CORSA ee 3 General Information and Safety a 8 Declaration of Conformity ll ll aaa 10 Explanation of Symbols 2200200200000 00 l0 ll a a aaa 11 Electrical Sale NAAN see an Aba and 12 S curit Electronique a 12 kasero AIO ah ec 13 S curit LAS sca etal ANDAS AD NAN 14 Biological Safety ce aah ee ese eel ee 15 S curit BiologiqueBiorisques 22 2 2 2 0 c ccc cece ec ceccecceccecceeceeeeeee 15 Spare Farts AY 15 Technology Overview aaa 18 LUI ER 20 Sterilizer Cleanser and Debubbler 2 20 SO Ossett gee eae ea 20 Sneak kaa man AY NANANA API E pas Ge BAGS ean ag HEAD 20 INSPIRE User Interface LL 21 The Image Gallery 2 22 Image Gallery Tools 0 002000 00 00 la 22 Image Display TOO NA ears a 22 The Analysis Area AA peng aoe ee eet eae 23 Analysis Area 008 wrk aa balana 2 2 NAL 23 Chapter 1 The Instrument Control Panel 0 0 020 2 c cece cece eee c ec eccececceceececeese 24 AA ee arene e tne ne eae een wear ere eet erent eee ae ee eek meee eer eae 28 Daily Operations 0000aaa 0000000 an0a 000000 oaoa 000000 aoaaa 00000 a a a20 00ra 22222 31 Turning on the ImageStreamX 2 31 Preparing to run and calibrating the ImageStreamX 2 2 2 2 22 2 31 Data Acquisition 0 000000000 aoaaa anaana
60. m 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 Securite Electronique Alimentation 100 240 V altenatif 50 60 Hz 3A Les hazards lectrique se trouvent dans l appareil surtout pres 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 a fond 12 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 Maximum Power 70 380 nm 0 or 85 mW 00 413 nm 50 mw 483 493 nm 200 mW or 400 mW high power option 58 562 nm 00 mW 92 593 nm 00 mW 35 647 nm 50 mw 20 740 nm O mW 75 800 nm 00 mW 15 840 nm 80 mW Sl NI No ala Bios Y lt D D gt The following laser warning label appears on the inside surface of the hood ADANGER Visible and or invisible Laser Radiation Avoid direct exposure
61. mes to the output files and parameters to include in a well plate report that is generated once the plate has completed While the plate is running the user is notified of any errors encountered via email The instrument can also sterilize at the completion of the plate Workflow e 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 e 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 e 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 e Run the plate see procedure below Access to AutoSampler operations is found under the AutoSampler menu 41 Chapter 3 From this menu you may e Create a plate definition e Runa plate CE e Define Plate e Run a single well from a plate Run Pete Extend or Retract the nest Exbend Hest Retret het To begin 1 Choose Define Plate from the Autosampler menu to open the Well Plate Def inition window Well Plate Definition Output Max Aci Filename Date Well Color File Path Time min Count Include in filename no no n no
62. meters Num Settings Channel OF Interest Num samples E Total Range Flow Core Axial Stability 0 10471 0 08 0 06 0 04 0 02 0 00077178 0 40 50 Sample Number Restoring Instrument Settings Database Setting Saved Value Result Value ASSIST ASSIST FlowSpeedCV 0 03 e AS515T Flow5peedCYMax 0 15 ASSIST FlowSpeedC PercentOverMax 0 10 Percent Over Max The flow core axial stability test plots 100 flow speed sample intervals each of which consists of an average velocity measurement of approximately 50 SpeedBeads thereby measuring the speed of approximately 5000 SpeedBeads The test computes a running average of all measurements which is listed under results on the pop up window and ensures that no more than 5 of all measurements exceed a 0 15 speed variation This ensures that synchronization is maintained between the imagery and the camera to better than a fraction of a pixel Test results 60 are stored in the ASSIST database The results and limits of the test are shown below the list when the test is selected 61 Chapter 4 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
63. mple 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 87 Chapter 5 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 lically 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 lin the fluidic system Run the purge ICS bubbles script from the instrument drop down menu See solutions for unstable fluidics Objects are rotat ing in the core stream Re run the focus adjuster and frame Objects are out of Camera line rate is offset calibration in ASSIST and verify focus incorrect it passes Excessive core stream variation due to air
64. nd analyzed to determine the intensity of each bead The median intensity for each position is then plotted and fit to a fourth order polynomial The peak height of the polynomial is then determined This position is the point where the peak intensity of the Gaussian laser beam intersects the center of the flow core This position provides both the highest intensity for illuminating the core stream and the point with the lowest coefficient of variation This position is stored for each laser and used as the default position during subsequent assays aa 488 Horizontal Laser Calibration Parameters Hum Settings 20 Channel OF Interest 1 Hum samples 400 w Total Range 1 O Ea ta TE o l Horizontal Laser Position um Idle Database Setting Saved Value Result Value ASSIST ExcHorizontalLaserPosilion 488 9 22 822 ASSIST ExcPowerl est_LaserPower 488 100 00 The result for the calibration are shown below the list when the calibration is selected 54 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 a Side Scatter 40x
65. ned 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 El Total Range nn SATNARSENSARNARENRNAESRRRAERENNENEARRRNES Restoring Instrument Settings Database Setting Saved Value Result Value AS515T FlowCorePositionT est FocusDeltaMax 15 00 Calculated Focus Position Delta AS5SI5T FlowCorePositionT est FocusPosYalue 219 16 pagasa AS515T FlowCorePositionT est_FocusDefault alue 217 50 ASSIST FlowCorePositionT est_LateralD eltaM ax 30 00 Calculated Lateral Position Delta The result and the limits for the calibration are shown below the list when the calibration is selected 63 Chapter 4 Focus Offset Test Measures the offset between the focus determined by the AFFS system and location of the peak response of the Image Collection system This test performs a pan through focus while simultaneously collecting SpeedBead focus data from the AFFS system and SpeedBead image data from the image collection system The AFFS data are processed to find the zero crossing point of no defocus and the image data are processed to determine the peak response point of highest spatial resolution Both sets of data are plotted as a function of Z position along the horizontal axis The AFFS zero crossing and image collection system peak response are indicated vertical li
66. nes 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 64 eo Focus Offset 40x Test Parameters Num Settings Num samples Total Range 0 6 0 5 0 4 0 3 0 2 Ensquared Energy Ratio 0 060156 M ee 4 2 Focus Score Channel Of Interest Image Collection l l 0 2 4 6 9 9 Focus Position um Autofocus l l 0 2 Focus Position um Idle Database Setting Saved Value Result Value AS515T FocusOffsetT est Value 0 60 ASSIST FocusOffsetT est LimitMin 0 50 A5515T FocusOffsetT est LimitMax 2 50 ASSIST SideScatterCalibPower 1 92 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 determine
67. o 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 the X Axis at 0 allows you to set the display pixel intensity to O 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 hannel 7 Minimum Pixel Intensity Maximum Pixel Intensity Automatic Manual Image Display Mapping X Ads Scale Set to Default Full Scale Set Range to Pixel Data Set Linear Curve Pacet All Sattinas 7 CA v 7 v J v i Ig Saturation Color Enable All Channels Reset All Colors 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 ob
68. 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 Turn 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 CES uncheck the EDF checkbox Lateral deviation of the core stream due to airorcloginthe 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 adjust focus and centering left or right until the images are centered andin optimal focus ii Incorrect Mag In the magnification and EDF section Autofocus and cen tering is not tracking properly 88 Symptom Possible Causes The two brightfield images are not of rect the same cell Illumination is grossly misaligned Cross correlation is incorrect Images appear pix elated or larger than normal Objects appear larger or smaller then normal Not all 12 channels are being dis played Image gallery zoom
69. ot fix the problem call Amnis service Index Acquisition button 25 stop 25 Acquisition button 25 Analysis Area 23 tools 23 ASSIST 47 ASSIST Calibrations 49 Autofocus S Curve 68 Brightfield crosstalk coefficient 53 camera synchronization 49 Dark Current 51 Horizontal laser 54 retro 56 side scatter 55 spatial offsets 50 ASSIST Tests 57 BF Intensity Selection 58 BF uniformity 59 Excitation laser power 57 flow core axial stability 60 flow core lateral stability 62 93 Chapter 6 flow core position 63 focus offset 64 image quality ensquared energy test 66 S Curve Peaks 75 ASSIST Utilities 68 brightfield calibration 70 EDF excitation 71 focus pan 73 PMT focus 74 Autosampler 41 Brightfield adjustments 26 34 Calibration 31 47 Brightfield Crosstalk Coefficient 53 Camera Synchronization 49 Dark Current 51 Horizontal Laser 54 Retro 56 Side Scatter 55 Spatial Offsets 50 Calibrations and Tests Start All 47 camera 18 94 Centering adjustment 27 Cleanser 20 Compensation button 23 mode 36 Core Tracking 32 data files 35 Debris Squelching 37 Debubbler 20 DNA dye cleaning 35 EDF characteristics 40 turing on 34 using 40 Features Area 34 Intensity 34 Max Gradient Intensity 34 Mean Pixel 34 95 Chapter 6 Raw Max Pixel 34 Raw Min Pixel 34 Saturation Count 34 Saturation Percent 34 fluorochrome balancing 33 Focus adjustment 27 Graphs features 34
70. ove field of view core track left or right to find the core are being over used Imaging and acqui Make sure the sample concentration is sition rate is Sample con between 10 and 108 cells ml Lower erratic or appears centration is low concentrations can be used but this frozen 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 no cells 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 Make sure the brightfield lamp is turned on and click Set Intensity 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 The sample is too concentrated 1 10 x10 7 cells per ml To decrease the event rate dilute the sample 86 Objects are not centered in the channel Camera is not track ing the cell velocity There is only one channel of bright Lateral deviation of the core stream due to air or clog in the system Autofocus and cen tering is not tracking properly Troubleshooting 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 sa
71. ple buffer concentration of these dyes so that the cells can be imaged properly Typ ically the concentrations in Current Protocols in Cytometry should work Instrument is expe or bright or riencing large tem changes over time perature variation Direct a fan toward the back of the instrument to dissipate excess heat or move the system to a temperature con trolled environment Laser power set too Increase or decrease the 785 SSC high or low laser power Core stream posi Run the purge bubbles script from the tion is grossly off instrument drop down menu See solu Allow the instrument to warm up by running for 15 minutes center within the tions for unstable fluidics 91 Chapter 5 Recommended Solutions flow cell due to air or clog in the fluid ICS Large variation in brightfield intensity levels Large flow speed variation due to air Light source deliv ering variable out put Brightfield intensity Intensity set before level sets incor desired flow speed rectly has been achieved Movable optics are out of position 92 Run the purge bubbles script from the instrument drop down menu See solu tions for unstable fluidics Power down and power up the instru ment if this does not fix the problem call Amnis service Allow the system to stabilize after loading a sample and then click Set Intensity Power down and power up the instru ment if this does n
72. re 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 Fill the Rinse Cleanser Sterilizer and Debubbler bottles if necessary Empty the Waste bottle Remove any tubes from the uptake ports 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 Bb WN 39 Chapter 3 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 BlueY EDF Excitation Utility on page 71 To collect a data file using the EDF element 1 2 3 4 Set instrument settings for the experiment Select EDF 1 from the collection f
73. ringe is Load sheath then go to the instrument empty drop down and run prime 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 Make sure the sample concentration is Sample con between 10 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 Insufficient illu Turn the appropriate lasers on Set the mination 785 SSC laser to 40mw Set the laser 79 Event rate slows Cells have settled in the lines Core is off center Chapter 5 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 10 bleach followed by a PBS wash to remove all traces of the DNA dye in the instrument or run the sterilize script 30min This suggests a minor clog Load a sam ple of 10 bleach followed by a PBS Cells are not dis played due to over clipping Cross con
74. s Total Range BF Uniformity E so a E o l l l 3 3 5 4 Camera Channel Idle Database Setting Saved Value Result Value ASSIST BFPeakVariation 0 921 020 SATO OSs KH ASSIST BFPeakVariationMin 0 00 0 00 0 ASSIST BFReakVarnationMax 1 50 1 50 1 ASSIST DarkCurrent0_Gene_2 22 205156 ASSIST DarkCurentl Genz 2 22 066406 ASSIST DarkCurent Gen2 2 23 039062 ASSIST DarkCurent3 Genz 2 24 253906 59 Chapter 4 Flow Core Axial Stability Test Measures the stability of the core stream velocity over time Measures the variation in the speed of the core stream as a percentage of the mean sample speed The ImageStream is designed to automatically sterilize cleanse and purge air from its fluidics systems after every day of operation Improper sterilization contaminants partially clogged fluidic lines air bubbles or non homogenous sheath solution can lead to excessive sample speed variation Although the ImageStream very accurately measures the sample speed to synchronize camera line rate with cell movement on the detector excessive speed variation can lead to small amounts of desynchronization The flow core axial stability test verifies that the fluidic system is operating within normal limits thereby providing the collection system with hydrodynamically focused objects traveling at a consistent speed for proper image synchronization bas Flow Core Axial Stability Test Para
75. s 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 e Layout menu 29 Chapter 3 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 e Compensation menu View edit or create a new compensation matrix View matrix Opens the compensation matrix view Edit matrix Opens the compensation matrix for editing New matrix Opens the compensation wizard e Advanced menu For field service personnel only Advanced Fluidics Autofocus Flow Speed Camera Iumination Acquisition Configuration Errar About ImageStream Access the current INSPIRE version number with the About ImageStream option 30 Daily Operations 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 turn the ImageStream on if the power is off Note If the ImageStream power is on and INSPIRE is already launched go directly to
76. s 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 Ea Brightfield XTalk Coefficient Calibration History 9425 2009 9 04 54 AM Parameters Num Settings Channel Of Interest Mum samples iF Total Range Channel ve BF Intensity pa D o un GB fe pi m o He m l 3 5 Camera Channel Database Setting Saved Value Result Value ASSIST DarkCurrentO_Gene_2 21 5039 21 ASSIST DarkCurrentl_Gene_2 21 5625 21 ASSIST DarkCurent Gen 2 2168 22 ASSIST DarkCurent3 Gen 2 23 082 22 ASSIST BFCrossT alkCoelfficientsChan1 1 00 0 01 O ASSIST BFCrossTalkCoefficientsChan 0 06 1 00 0 0 06 1 00 0 09 0 03 0 01 DM OOOO ASSIST BFCroseTalkCoefficientsChans 007 0 04 1 F 53 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 a
77. smitted light from the cells The collected light in optical space intersects with the spectral decomposition element Light of different spectral bands leaves the decomposition element at different angles such that each band is focused onto 6 different physical locations of one of the two CCD cameras with 256 rows of pixels As a result each cell image is decomposed into six 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 18 Chapter 3 Operating the ImageStream Using INSPIRE M 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 syst
78. step 4 1 Press the green power button inside the front door of the ImageStream to turn on the instrument and start the computer Log on with the user name Amnis and password is 100 Launch the INSPIRE software and by double clicking the INSPIRE icon on the desktop Preparing to run and calibrating the ImageStream 4 Fill the rinse bottle with deionized water and the sheath bottle with PBS Ensure the SpeedBead reagent is loaded on the bead port and is well mixed The beads are automatically mixed while the instrument is in use If the instrument has been idle for a long period remove the bead vial and vortex Refer to the following com patibility chart to choose the appropriate Sheath fluid WaterlSufactant Water Surfactant 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 10 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 t
79. sure that residual dye does not stain the subsequent samples 14 Change the file name for the next sample and continue collecting samples 15 Repeat for each sample 35 Chapter 3 16 When finished running all of the experiment samples run single color com pensation controls with the same laser settings as the experimental samples with the exception of the scatter laser 785 which turns off in compensation mode 17 Click ki 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 18 Follow the prompts in the wizard to collect all compensation control files 36 Data Acquisition Optional settings Setting the Image Display Properties 4 2 3 Click on in the acquisition section to open the window Select the channel by clicking on the channel name To change the channel name type a new name To change the channel color click on the color box To set the 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 t
80. t and pressing the start button in the popup window If the test fails three times in a row call Amnis service Focus adjustor cal Oo D bration failure Verify brightfield is working properly Frame Offset cal maa DS bration failure Verify brightfield is working properly Spatial Offsets cal a ibration failure Verify brighttield is working properly Make sure the excitation lasers are off Dark Current cal jand brightfield is blocked Completely ibration failure 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 Horizontal Laser power properly Completely power down Calibration failure jthe 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 Side Scatter Cal can set power properly Completely bration failure power down the instrument and power back up to re run the test Verify spatial offsets passed Retro Calibration failure 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 Brightfield align ae RP ENT Verify brightfield is working properl
81. t 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 LISLITITLIILATATETALILILILEILEILILISELILILIGLITITITALITEITILELAITITLILELEILILISELILILISELILILILEILEILILISELILLLELLLLL D CLARATION OF CONFORMIIY IN ACCORDANCE TO ISO IEC GUIDE 67 FOR A Cellular Analyzer MANUFACTURER Amnis Corporation 645 Elliott Avenue W Suite 100 Seattle WA 98119 Phone 206 576 6865 MODEL NUMBER ImageStream X Mk II REPORT 2 582624 02 and FILQ3326 025 DIRECTIVES EMC Directive 2004 108 EC amp Low Voltage Directive 2006 95 EC STANDARDS Flectncal Equipment for Measurement Control and Laboratory Use EMC Re Part 1 General Requirements EN 61326 1 2006 edition Indusinal Scientific and Medical Equipment Radio Frequency Disturbance Charactenstics Limits and Methods of Measurement CISPR 11 2009 edition Information Technology Equipment Radio Disturbance Characteristics Limits and Methods of Measurement EN 55022 1998 CISPR 22 1997 edition Electromagnetic Compatibility EMC Part 3 2 Limits Limits for Harmonic and Emissions Equipment Input Current 16 A per phase IEC 61000 3 2 2009 edition EMC Part 3 3 Limits es of
82. the Calibration window below 11 Click SaveDB to save the kernels to the database 12 Close the calibration window 7D Focus Pan Utility The Focus Pan Utility allows the user to define an automatic focus pan to be performed The user can enter the pan range in microns the number of intervals within the pan and the number of samples to be taken per interval The utility can be used in conjunction with the Run Acquire mode to capture and image file throughout a focus pan This test particularly helpful to service personnel for the process of characterizing the performance of the instruments Extended Depth of Field capability The utility is not run as part of the routine ASSIST tests bas Focus Pan Utility Parameters Num Settings 5 Channel Of Interest Num samples 1 000 a Total Range 4 Acquisition Progress pr o E 3 2 QC wn Focus Position um Database Setting Saved Value Result Value 73 Chapter 4 PMT Focus Utility The PMT Focus Utility provides service personnel with visual and quantitative feedback to characterize the physical alignment and gain settings for each channel of the AutoFocus and Flow Speed system The utility pans the focus stage over a 40 micron range while collecting data at one micron intervals from each channel of the AFFS system The data peaks are determined and fit to 2nd order polynomials to provide a quantitative characterization of the Grating spacing and signal levels for
83. to beam The following laser warning label appears on the interior side panels near release mechanisms and next to hood latch release Caution Using controls making adjustments or performing procedures other than those specified in this manual may result in hazardous exposure to laser radiation 13 Chapter 1 Securite Laser L ImageStreamX c est une appareil au laser Classe qui se conforme a 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 815 840 nm 180 mw Les etiquettes d avertissement suivantes sont place s dans l interior du capot DANGER slble et Invisible radiations laser quand ouverte et ks enclechemernts sont faites Exposition dangereuse au rayonnement direct ou diffus pour lical et amp peau Laser Classed dans l appareil keer Classe 1 Les etiquettes d avertissement suivantes sont placees dans L Interieur de panneaux lateraux pres de mecanismes de liberation et a cote du loquet de fermeture de capot Avertissement L utilisation des commandes ou les rendement des procedures autres que celle precisees aux presentes peuvent provoquer une radioexposition dangereuse 14 Biological Safety Biological Safety Biohazards The Image
84. ube 7 Click Start All Calibrations and Tests in the calibration window 31 Chapter 3 8 Center the core stream images if necessary by laterally moving the objective under Focus and Centering Core Tracking is adjusted by pressing right or left arrows to center images 9 The event rate should be 100 300 beads 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 Troubleshooting on page 77 or call your Amnis Field Service Representative For more information on the individual calibrations and tests refer to theFigure ASSIST Calibrations on page 49 in chapter 4 10 When the calibrations and tests have passed close the Calibrations window 392 Data Acquisition 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
85. udes the ImageStream multispectral imaging flow cytometer and the INSPIRETM and IDEAS TM 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 17 Chapter2 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 ImageStream CCD camera allows up to 1000 times more signal to be acquired from cells in flow than from conventional frame imaging approaches Velocity detection and autofocus systems maintain proper camera synchronization and focus during the process of image acquisition The following diagram illustrates how the ImageStreamX works detector velocity detector autofocus cells in flow 4 brightfield illuminator Hydrodynamically focused cells are trans illuminated by a brightfield light source and orthogonally by a laser s A high numerical aperture NA objective lens collects fluorescence emissions scattered and tran
86. voltage changes voltage fluctuations and flicker m public low voltage supply systems for equipment with rated current lt 16 per phase and not subject to conditional connections IEC 61000 3 3 1995 Electtomagnetic 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 Immmumity Test EN 61000 4 3 2008 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 4 Electncal Fast Transient Burst Immunity Test EN 61000 4 4 2004 edition Electtomagnetic Compatibility Part 4 Testing and Measurement Techniques Section 3 Surge Immmmity Test EN 61000 4 3 2005 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 6 Immunity to Conducted Disturbances Induced by Radio Frequency Fields EN 61000 4 6 2008 edition Electtomagnetic Compatibility Part 4 Testing and Measurement Techniques Section Power Frequency Magnetic Field Immmmuty Test EN 61000 4 8 2001 edition Electromagnetic Compatibility Part 4 Testing and Measurement Techniques Section 11 Voltage Dips Short Interruptions and Voltage Vanations Immmumity Test EN 61000 4 11 2004 edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory
87. werl est LaserPower 488 100 00 ASSIST AetroCalibCenterPosition 620 00 56 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 collection path The test compares the mean signal strength acquired from each laser and compares it to radiometric ally calibrated signal strengths collected during the manufacturing process The intensity of each laser is stored in the database The results and limits of the test are shown below the list when the test is selected 57 Chapter 4 BF Intensity Selection Test Verifies the BF intensity calibration for each BF mode The image
88. y 81 Laser Power test failure Chapter 5 Possible Causes Recommended Solutions Brightfield uni pes 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 Camera noise test failure for unstable fluidics Flow Core Lateral See solution above Flow core Axial sta Stability test failure bility test Flow Core Position See solution above Flow core Axial sta test failure bility test Focus Percentage See solution above Flow core Axial sta test failure bility test Focus Uniformity See solution above Flow core Axial sta test failure bility test 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 mog thai SI GE 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

INSPIRE

Contents

Download Pdf Manuals

Related Search

Related Contents