Amnis ImageStreamX MKII imaging flow cytometer User's Manual
The Amnis ImageStreamX MKII 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 ImageStreamX MKII combines the per cell information content provided by standard microscopy with the statistical significance afforded by large sample sizes common to standard flow cytometry.
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INSPIRE
®
ImageStreamX® System Software
User's Manual
Part Number: 780-01286-01 Rev: B
For updates log in at www.amnis.com
Patents and Trademarks
Amnis technologies and products are protected under one or more of the following U.S. patents: 6211955; 6249341; 6256096; 6473176; 6507391;
6532061; 6563583; 6580504; 6583865; 6608680; 6608682; 6618140; 6671044;
6707551; 6763149; 6778263; 6875973; 6906792; 6934408; 6947128; 6947136;
6975400; 7006710; 7009651; 7057732; 7079708; 7087877; 7190832; 7221457;
7286719; 7315357; 7450229; 7522758, 7567695, 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. EMD Millipore is a division of Merck KGaA, Darmstadt, Germany.
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
Table of Contents
General Information and Safety
Chapter 2: Introduction to the ImageStreamX®
Chapter 3: Operating the ImageStreamX® Using INSPIRETM
Sterilizer, Cleanser, and Debubbler
Setting the Image Display Properties
Preparing to run and calibrating the ImageStream
Loading and running the sample:
Collecting and saving the data files
Setting ImageStreamX Speed and Sensitivity
Chapter 4: Instrument Calibrations and Tests
Chapter 1: Information and Safety
Chapter 1: Information and Safety
This section covers safety information for operating the Amnis
ImageStream
X® multispectral imaging flow cytometer. Anyone who operates the ImageStream
X should be familiar with this safety information. Keep this information readily available for all users.
The safety information consists of the following areas:
•
General Information and Safety
•
•
•
•
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Chapter 1
General Information and Safety
The ImageStream
X imaging flow cytometer is manufactured by Amnis Corporation and has a rated voltage of 100–240 VAC, a rated frequency of 50/60 Hz, and a rated current of 3 A. The years of construction were 2004–2014, 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 o
C through 25 o
C; and at a maximum relative humidity of 80%, non-condensing. During instrument operation the ambient temperature should be maintained within +/- 2 o
C. The mains power supply may not fluctuate more than +/– 10% and must meet transient over voltage category (II). The instrument is evaluated to Pollution Degree 2.
Noise level: The noise level of the ImageStream
X 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
X relies on many delicate alignments for proper operation. The machine may be moved only by an Amnis representative.
Cleaning: Clean spills on the instrument with a mild detergent. Using gloves clean the sample portal and sample elevator with a 10% bleach solution. Dispose of waste using proper precautions and in accordance with local regulations.
Preventative maintenance: The ImageStream
X 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
X is intended only for Amnis service personnel.
Protection impairment: Using controls or making adjustments other than those specified in this manual can result in hazardous exposure to laser radiation, in exposure to biohazards, or in injury from the mechanical or electrical components.
FCC compliance: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to part 15 of the FCC rules. These limits
- 2 -
Chapter 1: Information and Safety were designed to provide reasonable protection against harmful interference when the equipment is used in a commercial environment. This equipment generates, uses, and can radiate radio-frequency energy and, if not installed and used in accordance with the instruction manual, can cause harmful interference to radio communications. The operation of this equipment in a residential area is likely to cause harmful interference—in which case the user will be required to correct the interference at the user’s own expense.
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Chapter 1
Declaration of Conformity
- 4 -
Chapter 1: Information and Safety
Explanation of Symbols
Label
Table 1:
Location
Waste tank
Power supply cover
Hazard
Risk of exposure to transmissible biological disease.
Risk of injury by electric shock.
Power supply Protective earth ground.
Inside surface of hood
Risk of exposure to hazardous laser radiation.
Interior, side panels near release mechanisms and
Risk of exposure to next to hood latch release hazardous laser radiation.
On the back of the instrument
No laser radiation is accessible to the user during normal instrument operation.
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Chapter 1
Electrical Safety
Equipment ratings: The ImageStream
X 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.
Sécurité Electronique
Alimentation: 100–240 V altenatif, 50/60 Hz, 3A.
Les hazards électrique se trouvent dans l’appareil, surtout prés de la source d’alimentation. Pour éviter les choks électriques, introduire la lame le plus large de la fiche dans la borne correspondante de la prise et pousser à fond.
- 6 -
Chapter 1: Information and Safety
Laser Safety
The ImageStream
X 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
X may have the following lasers:
Wavelength
370-380 nm
400-413 nm
483-493 nm
558-562 nm
592-593 nm
635-647 nm
720-740 nm
775-800 nm
815-840 nm
Table 1:
Maximum Power
30 or 85 mW
150 mW
200 mW or 400 mW (high power option)
200 mW
300 mW
150 mW
50 mW
100 mW
180 mW
The following laser warning label appears on the inside surface of the hood:
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.
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Chapter 1
Sécurité Laser
L'ImageStreamX c'est une appareil au laser, Classe I, qui se conforme à 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:
Longueurd’opnde
370-380 nm
400-413 nm
483-493 nm
558-562 nm
592-593 nm
635-647 nm
720-740 nm
775-800 nm
815-840 nm
La Puissance Maximale
30 or 85 mW
150 mW
200 mW or 400 mW (high power option)
200 mW
300 mW
150 mW
50 mW
100 mW
180 mW
Les etiquettes d’avertissement suivantes sont placeés dans l’interior du capot:
Les etiquettes d’avertissement suivantes sont placeés dans L'Intérieur, de panneaux latéraux près de mécanismes de libération et à côté du loquet de fermeture de capot.
Avertissement: L’utilisation des commandes ou les rendement des procedures autres que celle preciseés aux presentes peuvent provoquer une radioexposition dangereuse.
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Chapter 1: Information and Safety
Biological Safety
Biohazards: The Image Stream is rated at BSL1. Do not load or flush samples containing infectious agents without first exposing the sample to inactivating conditions. It is recommended that samples be fixed in 2% paraformaldehyde for at least 10 minutes before running the samples on the ImageStream
X
.
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 biohazardhandling 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.
Sécurité BiologiqueBiorisques:
L'image Stream est évalué à un niveau de sécurité biologique L1. Ne pas acquérir ou vider des échantillons contenant des agents infectieux sans les avoir inactivés. Il est recommandé que les échantillons soient fixés dans du paraformaldéhyde 2% pendant au moins 10 minutes avant d'acquérir des échantillons avec l’ImageStreamX.
L'utilisation, le confinement et l'élimination des matériels biologiques dangereux sont tenus d'être en conformité avec les normes de sécurité relatives au laboratoire et de la directive 93/95/E et restent sous la responsabilité de l'utilisateur. Respectez la réglementation en vigueur pour le traitement et l'élimination des déchets dans des réservoirs prévus à cet effet.
Prévenir l'accumulation des déchets en vidant le réservoir lorsque l'indicateur indique qu'il est plein. Stériliser les instruments de routine après chaque journée d'utilisation. Notez que cette procédure ne garantit pas la stérilité vis à 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.
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Chapter 2: Introduction to the ImageStreamX®
Chapter 2: Introduction to the
ImageStream
X®
The Amnis ImageStream
X 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
X 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
X system, fluorescence intensity measurements are acquired as with a conventional flow cytometer; however, the best applications for the ImageStream
X 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
X system includes the ImageStream
X imaging flow cytometer and the INSPIRE
TM and IDEAS
TM multispectral software applications.
The INSPIRE software is integrated with the ImageStream
X and is used to run the instrument. INSPIRE also provides tools for configuring the ImageStream
X
, defining cell parameters, and collecting data files for image analysis. The IDEAS software is used for spectral compensation, image analysis and statistical analysis of the images acquired by the ImageStream
X multispectral imaging flow cytometer.
Technology Overview
The ImageStream
X 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
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Chapter 2 used by the ImageStream
X
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.
Hydrodynamically focused cells are trans-illuminated by a brightfield light source and orthogonally by laser(s). A high numerical aperture (NA) objective lens collects fluorescence emissions, scattered and transmitted light from the cells. The 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.
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Chapter 3: Operating the ImageStreamX® Using INSPIRETM
Chapter 3: Operating the
ImageStream
X®
Using INSPIRE
TM
This chapter describes the operation of the ImageStream
X system using the INSPIRE software. Daily operation involves an initial calibration and testing of the system using SpeedBeads and
ASSIST, followed by sample runs and data acquisition, and finally sterilization of the system to prepare for use the following day.
Optimizing instrument setup for sample runs is also described here in detail.
•
•
•
•
•
•
- 13 -
Chapter 3
Fluidics
Sterilizer, Cleanser, and Debubbler
These recommended reagents have been formulated to optimize the performance of the ImageStream
X 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
Cleanser
Debubbler
Sterilizer
Name
Coulter Clenz®
70% Isopropanol
0.4-0.7%
Hypochlorite
Source*
Beckman
Coulter
Millipore
VWR
PBS, Ca
++
Mg
++ free Millipore
deionized water
Catalog #
8546929
1.37040
JT9416-1
BSS-1006-B
(1X)
6506- (10X)
Sheath
Rinse
* 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
X
, 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.
Sheath Fluid
Two bottles are provided: one labeled Sheath to be filled with phosphate buffered saline (PBS with no surfactants) for running samples and one labeled Rinse to be filled with de-ionized (DI) water for rinsing the instrument during shutdown. Fluid is drawn from these bottles into the sheath and flush syringe pumps. The sheath pump controls the speed of the core stream and the size of the core stream diameter. The flush pump is used to clean and flush the system and alternating with the sheath pump also controls the core.
- 14 -
INSPIRE User Interface
INSPIRE User Interface
The user interface is divided into 3 areas, the image gallery where channel images are displayed, a work area where graphs of features are displayed and the controls section where the instrument is controlled.The layout of the Image
Gallery and Analysis area can be vertical or horizontal and changed under the
Layout menu. Status information is displayed along the bottom of the window.
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Chapter 3
The Image Gallery
Images are displayed in the image gallery during setup and acquisition.
Image Gallery Tools
Icon Name
All
Description
Select the population to view
Pause Pause/Resume the display
Up/Down Move up or down in the image gallery while paused
Zoom in Enlarges the imagery
Zoom out Resets the zoom
Display Opens the display settings window
Mask Displays the segmentation mask on the images
Color Turns pseudocolor ON/OFF wrench Tools to measure pixel intensity of displayed images channel name
Click to change selection of channels to view/acquire
Setting the Image Display Properties
There are two methods for setting the display of the imagery. The display properties wizard will adjust the display as the images are being collected. The display can also be set manually by the following procedure. Note that these settings adjust the monitor display and do not change the pixel intensities of the images collected.
Using the wizard
1 Click on the wand next to the analysis area tools and select the Display
Settings Wizard.
2 Select the channels to set and click Finish.
The wizard will adjust the display for a channel with greater than 75 Raw Max
Pixel values using the Min and Max pixels from the images being collected. If
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INSPIRE User Interface there are no positive events in a channel the default settings (35-750) are used.
The manual settings may be used for channels with rare events since there may not be enough data for the wizard.
Manual method
1 Click on the Display Settings tool to open the window.
2 Select the channel by clicking on the channel name.
3 To change the channel name, type a new name. To change the channel color click on the color box.
4 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 to the adjustments can be visualized in the image gallery.
At each intensity on the X Axis of the graph, the gray histogram shows the number of pixels in the image. This histogram provides you with a general sense of the range of pixel intensities in the image. The dotted green line maps the pixel intensities to the display intensities, which are in the 0–255 range.
Manual setting is done by Click-dragging the vertical green line on the left side
(crossing the X Axis at 0) allows you to set the display pixel intensity to 0 for all intensities that appear to the left of that line. Doing so removes background noise from the image.
Click-dragging the vertical green line on the right side allows you to set the display pixel intensity to 255 for all intensities that appear to the right of that line.
If a non-linear mapping occurs by moving the yellow crosshair, click 'Set Linear
Curve' to return to a linear transformation.
Note: Changing the display properties does not change the pixel intensity data.
They are for display purposes only.
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Chapter 3
Image Display Tools
• Ptr, Line, Rgn: Buttons that allow interrogation of pixel information of a single point (Ptr), a line, or a region (Rgn) of the imagery.
• Pixel Information box: Displays the selected Pixel (x,y) coordinates and its
Intensity value.
• Region of Interest box: Displays the Minimum, Maximum and Mean pixel intensity values, their standard deviation (Std. Dev.), and the Area of the drawn region.
• Intensity Profile: Plot of horizontal pixel number vs. Mean pixel intensity for the drawn region.
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INSPIRE User Interface
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 analysis area is the same as in IDEAS. Refer to the IDEAS user manual for further information on graphs, regions and populations.
Analysis Area Tools
Icon Name
Reset
Histogram
Scatter Plot
Description
Refreshes the graphs with incoming data
Opens the histogram graph tool
Opens the bivariate scatter plot tool
Pointer
Line region
Reset cursor to pointer
Draw a line region on a histogram
Rectangle region Draw a rectangular region on a scatterplot
Oval region Draw an oval region on a scatterplot
Polygon region Draw a polygon region on a scatterplot
Select All Selects all plots in analysis area
Tile
Size Plots
Tiles the graphs in the analysis area to fill the space
Sets size of selected plots to small, medium or large
Populations
Regions
Wizards
Opens the population manager
Opens the region manager
Opens the list of wizards
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Chapter 3
The Instrument Control Panel
The instrument control panel provides tools to control instrument operation, data acquisition and status.
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Filename
Seq#
Count of
INSPIRE User Interface
In the Sample section you can load a sample or return a sample.
Sample time remaining is displayed when a sample is running.
Loads the sample
Returns the sample
In the File Acquisition section you can type in a custom filename, set the sequence #, choose the data file folder, type the number of events and choose the population to collect.
Begin Acquisition
Type the filename
Choose the beginning sequence number
Navigate to the folder to save the data
Enter the number of events to collect in the box.
Choose the population to collect in the dropdown menu.
Collect a different population while counting the first population. Click on the bracket to break the link and select population.
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Chapter 3
In the Illumination section you can turn laser and brightfield illumination on or off and set intensities.Select the scatter channel, either 6 or 12. All lasers have variable power and are defined by their excitation bandwidth.
405nm laser excitation - currently set to OFF and 0 mW of power.
488nm laser excitation- currently set to
ON at 60 mW of power.
642nm laser excitation- currently set to
ON at 150 mW of power.
SSC (side scatter) laser excitationcurrently set to OFF at 2.00 mW of power. SSC is produced from a dedicated 785 nm laser.
Brightfield illumination is shown as ON in channels 1 and 9.
Sets the Intensity of the brightfield to
800 counts.
Select the magnification. Note: this is optional equipment.Turn EDF on by checking the box.
Adjust the speed and sensitivity for the run. (see table below for an explanation of speed versus sensitivity)
Run fluidics.
Stop fluidics.
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INSPIRE User Interface
Speed and Sensitivity are inversely related.
See the table below for more information of speed, sensitivity and resolution.
Focus and Centering can be adjusted using the right and left arrows.
Runs the startup script.
Runs the shutdown script and sterilizes the system.
Speed versus Sensitivity
The table below shows the pixel resolution, relative sensitivity and approximate time to collect 10,000 obects at a concentraion of 1 x 10
7 objects per ml at different magnifications and speed settings. In order to run at a higher speed the rows on the camera are binned, this means the signal is collected in 1, 2 or 4 rows of the
CCD at a time.
Note: Binning refers to the number of pixel rows used to collect the data.
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Chapter 3
Bottom task bar:
Status buttons are displayed at the bottom of the INSPIRE window.
Describes the current script
Level indicator for pumps
Green indicates compensation is being applied to the Intensity feature. Note that imagery and other features are not compensated.
Yellow- calibrations and tests not run
Red- one or more calibrations or tests failed
Green- all calibrations and tests have passed
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INSPIRE User Interface
Menu Bar
The menu bar is located in the upper-left portion of the INSPIRE screen. It consists of these four menus:
• File menu: Load and save instrument setup templates. A template contains instrument settings that can be predefined and loaded to simplify the instrument setup process.
— 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.
Template 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.
• Instrument menu: Run the ImageStream
X camera and instrument-specific fluidic scripts (automated fluidic routines).
— Start/Stop Camera:: Starts or Stops imaging.
— Calibrate with ASSIST: Opens the Calibrations and Tests window.
- 25 -
Chapter 3
— Load Sheath: Fills the sheath syringe with sheath fluid and an air bubble that facilitates stable flow.
— Flush Load Beads: Flushes the bead syringe and reloads beads from the bead tube.
— Load Flush Syringe: Fills the flush syringe with sheath fluid.
— Prime: Pushes sample and beads into the flow cell.
— Purge Bubbles: Removes air bubbles from the flow cell by filling the flow cell with air then filling the sheath line and pump with debubbler and rinsing the flow cell. The sheath syringe is then refilled with sheath and the bubble trap, lines and flow cell are filled with sheath.
— Purge Sample Load Line: Flushes the sample load line with debubbler to remove bubbles formed during sample loading.
— Abort Task: Stops the current script.
— View Tank Levels:: Opens the fluid level window.
— Service Scripts:: For field service personnel only.
— Options::
• Autosampler menu: Access autosampler controls.
— Eject Tray: Opens the door of the autosampler and extends the tray for the
96 well plate.
— Load Tray: Retracts the plate tray back into the instrument and closes the door.
— Define Plate: Opens the plate definition dialog.
— Run Plate: Starts the autosampler run as defined by the plate definition.
— Load From Well: Allows a single sample load from a well plate.
- 26 -
INSPIRE User Interface
—
• Analysis menu: Access the Feature, Population and Region Managers.
Functionality is the same as for IDEAS. Refer to the IDEAS user manual for more information.
— Features: Opens the Feature Manager. Features can be renamed or new combined features can be created.
— Populations: Opens the Populations Manager. View,edit or delete populations.
— Regions: Opens the Regions Manager. View, edit or delete regions.
Note: See IDEAS User manual for more information.
• Compensation menu: View, edit or create a new compensation matrix.
— Create Matrix: Opens the compensation wizard.
— Load Matrix: Applies compensation to the Intensity features.
— View Matrix: Opens the compensation matrix values table.
— Clear Matrix: Stops applying compensation of Intensity features.
• Layout menu:
— 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 separator between the image gallery and analysis area when images are added or removed from the view.
• Advanced:menu: For field service personnel only.
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Chapter 3
— About ImageStream
X
: Access the current INSPIRE version number with the About ImageStream
X option.
—
- 28 -
Daily Operations
Daily Operations
Turning on the ImageStream
X
:
This section describes how to prepare the ImageStream
X for use. The
ImageStream
X is usually left on with INSPIRE launched, but the following instructions also describe how to turn the ImageStream
X on if the power is off.
Note: If the ImageStream
X power is on and INSPIRE is already launched, go directly to step 4.
1 Press the green power button inside the front door of the ImageStream
X to turn on the instrument and start the computer.
2 Log on with the user name (Amnis) and password (is100).
3 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 compatibility chart to choose the appropriate Sheath fluid.
Sample Solution
PBS
PBS
PBS/Surfactant
Sheath Fluid
PBS
Water
PBS
Acceptable
Yes
Yes*
Yes
PBS/Surfactant
Water
Water
Water/Surfactant
Water
PBS
Water
PBS
No
Yes
Yes
No
Water/Surfactant Water
* Cells in PBS run with water sheath will swell.
Yes
5 Empty the waste tank. Push on the quick-disconnect buttons to remove the tubing from the waste tank. Add 160 ml of bleach to the Waste bottle. The final volume 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.
6 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 µl of air into the uptake line. Beads are loaded into the bead pump from the 15 ml conical tube. The Initializing fluidics window opens showing the progress bar.
The box is checked by default to run ASSIST after the fluidics are ready.
Uncheck the box if you wish to start ASSIST later.
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Chapter 3
7 Click Start All Calibrations and Tests in the calibration window if ASSIST was not run in the previous step.
8 Center the core stream images (if necessary) by laterally moving the objective under Focus and Centering. Centering is adjusted by pressing right or left arrows to center images.
9
The event rate should be 800-1000 events per second. (If not, see Chapter 5:
10 When the calibrations and tests have passed the ASSIST status light will change to green. Close the Calibrations window.
11 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,
Note:
If adjustments are made to the instrument in order to pass an individual
ASSIST calibration or test, it is important to re-run all calibrations and tests in order to record the current settings after adjustments.
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Data Acquisition
Data Acquisition
After the ImageStream
X system is calibrated, you are ready to acquire experiment data files. The sample is loaded into the sample pump. Beads and sample are injected into the flow cell to form a single core stream that is hydrodynamically focused in front of the imaging objective. The beads are used by the system to keep the autofocus and camera synchronized during the sample run, while the objects from the sample are saved to the data file. To use the Autosampler for unattended
operation see Using the Autosampler.
Refer to the
ImageStream Sample Preparation Guide
for experimental set-up recommendations. Use compatible sample solutions from the table below.
Sample Solution
PBS
PBS
PBS/Surfactant
PBS/Surfactant
Water
Water
Water/Surfactant
Water/Surfactant
Sheath Fluid
PBS
Water
PBS
Water
PBS
Water
PBS
Water
Acceptable
Yes
Yes*
Yes
No
Yes
Yes
No
Yes
Sample order:
Samples from an experiment are typically run in the following order:
• Experimental sample with the brightest stains to set the sensitivity for the run
• 10% bleach to wash out DNA dye followed by PBS
• Using the compensation wizard or manually setting parameters
— Single color fluorescence controls (no DNA dye) NO BF or SSC
— Single color DNA dye control NO BF or SSC
• 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 In the file menu, choose Load Default Template.
Note: Application-specific instrument settings can be saved in a template and used to facilitate instrument setup, but it is recommended that you verify the appropriateness of the settings for the specific experimental run. The default template has a set of Raw Max Pixel scatterplots for all channels helpful for setting laser powers.
2 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 experiment once established on this sample.)
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When prompted place sample vial with 20-200 ul into the sample loader.
3 Choose the objective under Magnification (optional)
4
Select EDF collection if desired. See Using EDF for details.
5 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.
6 Select the channels to be collected by clicking on a channel name.This opens a window where channel selection can be set by checking the box next to the channel name.
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Data Acquisition
.
7
See Setting the Image Display Propertiesfor Setting the Image Display.
8 Select Brightfield channels. Default is Ch1 for a 6 channel system; Ch1 and 9 for a 12 channel system. Click Set Intensity.
9 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 following features in any channel are available:
• Area: The number of pixels in an image reported in square microns.
• Aspect Ratio: The Minor Axis divided by the Major Axis is a measure of how round or oblong an object is See below for the definitions for Major and Minor
Axis.
• Background Mean: The average pixel intensity of the background pixels.
• Gradient RMS: The average slope spanning three pixels in an image. This feature measures image contrast or focus quality.
• Intensity: The integrated intensity of the entire object image; the sum of all pixel intensities in an image, background subtracted.
• Major Axis:The longest dimension of an ellipse of best fit.
• Mean Pixel:The average pixel intensity in an image, background subtracted.
• Minor Axis:The shortest dimension of an ellipse of best fit.
• Object Number:The serial number of an object.
• Raw Centroid X:The center of the object in the X dimension of the frame.
• Raw Centroid Y:The center of the object in the Y dimension of the frame.
• Raw Max Pixel:The intensity value of the brightest pixel in an image (no background subtraction).
• Raw Min Pixel: The intensity value of the dimmest pixel in an image (no background subtraction).
• Time: The object's time value in seconds.
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• Uncompensated Intensity: The integrated intensity of the entire object image; the sum of all pixel intensities in an image, background subtracted.
See the IDEAS User Manual for more details on features and graphing.
Collecting and saving the data files
Once the sample is running and the ImageStream
X is properly set up, you are ready to acquire the data as a raw image file (.rif) and/or an FCS file. The .rif
contains uncompensated pixel data along with instrument settings and ASSIST information in a modified TIFF format. The file includes only those objects defined by the population selected in the acquisition section.
10 Enter the file name.
The number in the Sequence # box is appended to the file name, followed by the
.rif or .fcsextension. The sequence number increases by 1 with each successive data acquisition. Files collected with BF off will be appended with noBF and files collected 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: \,/,:,*,<,>, or |.
Note: to collect a .fcs file at the same time as a .rif file choose the .fcs option under the File menu.
11 Browse to select an existing folder or to create a new folder in which to save the files.
12 Enter the number of cells you want to acquire in the box after Count and select the population from the dropdown. To count one population but limit the collection to another population, click on the bracket to break the link and enable the Collect box.
13 Click on the Acquire button to begin acquiring a data file:
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Data Acquisition
14 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 discard the acquired data or to save the collected data in a file. The acquisition can be paused and resumed by clicking .
15 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 dyestained sample, Load a solution of 10% bleach and then Load PBS to ensure that residual dye does not stain the subsequent samples.
16 Change the file name for the next sample and continue collecting samples..
17 Repeat for each sample.
18 When finished running the experiment samples or after setting the template, run single color compensation controls with the same laser settings as the experimental samples with the exception of the scatter laser 785 which turns off in compensation mode.
19 Click in the analysis tools section and choose Compensation to begin
The compensation wizard will set up the instrument for compensation controls that must be collected with brightfield and scatter (785 nm laser)
OFF and every channel to be collected. Keep all laser powers the same as for the experimental samples.
Follow the prompts in the wizard to collect all compensation control files:
• Click Load or if a compensation control sample is already running, click Next.
• Place the tube on the uptake port and Click OK.
• Click Next when sample is running.
• Verify the channel for compensation.
• Draw a region on the Uncompensated Intensity scatter plot if not all cells are positive to define the positive population. View the population in the Image
Gallery and choose this population to collect.
• Name the file and choose the path to save the data.
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• Click Collect File. The compensation coefficients are calculated. The compensation coefficients and an Intensity scatter plot using the coefficients is displayed.
• Click OK on the Acquisition Complete popup window.
• Click Load to continue with the next single color control sample or click
Return (optional).
• Repeat the previous steps for each compensation sample. For each sample reset the Image gallery population to view All and then create an appropriate population for each sample. Note, the R1 gate can be moved for each sample.
• Click Exit when done and Save the coefficients to a compensation matrix file.
The beginning template is restored and the saved matrix is used to compensate the Intensity feature. Note that other features are not compensated. The compensation can be cleared if desired from the compensation menu. Scatter plots can be made with the feature Uncompensated Intensity to compare with and without compensation.
20 Continue to collect experimental files.
21 Click Shutdown when done. See
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Data Acquisition
Optional settings
Squelching Debris
Some samples have an abundance of small particulate debris. These can be eliminated from collection by gating or by using Squelch to reduce the sensitivity of object detection. As opposed to gating debris away from cells, squelching debris can prevent INSPIRE crashes related to overburdening the computer processor with an abnormally high event rate. Squelch should only be used if the rate of total objects per second reaches 4000. Squelch values range from 0 to 100; increasing the value decreases object detection sensitivity.
1 Choose All in the image gallery.
2 Observe the relative proportion of cell to debris images appearing in the imaging area and the event rate (Total/Sec under Acquisition Status).
3 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 µm at 40 X magnification. In order to collect images at higher speed, the rows on the camera can be binned. Image collection speed is inversely related to image resolution (sensitivity).
The table below shows the pixel resolution, relative sensitivity and approximate time to collect 10,000 obects at a concentraion of 1 x 10
7 objects per ml at different magnifications and speed settings. In order to run at a higher speed the rows on the camera are binned, this means the signal is collected in 1, 2 or 4 rows of the CCD at a time.
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Chapter 3
Note: Binning refers to the number of pixel rows used to collect the data.
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Daily Shutdown Procedure
Daily Shutdown Procedure
This procedure sterilizes the system and leaves it with pumps empty and water in the fluidic lines. The instrument is left on with INSPIRE running.
1 Fill the Rinse, Cleanser, Sterilizer, and Debubbler bottles if necessary.
2 Empty the Waste bottle.
3 Remove any tubes from the uptake ports.
4 Click Shutdown
Note: This procedure automatically turns off all illumination sources and rinses the entire fluidic system with water, sterilizer, cleanser, de-bubbler, and then water again. The sterilizer is held in the system for ten minutes to ensure decontamination. It takes about 45 minutes of unattended (walk-away) operation to complete.
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Chapter 3
Optional upgrades
Using EDF
Extended depth of field (EDF) is a novel technique used in a variety of applications including FISH spot counting where having the entire cell in optimal focus is critical to obtaining accurate results.
First images are acquired with the EDF element in place. The data is automatically deconvolved using the EDF kernel from the calibration prior to analysis using IDEAS. Calibration of the element is done by the Amnis engineer wheninstalled and should be repeated by Amnis service when any optical changes are made to the instrument.
To collect a data file using the EDF element
1 Set instrument settings for the experiment.
2 Check the box 'EDF' in the instrument control panel.
3 Adjust any regions being collected to accommodate using EDF.
4 The calibration kernels saved during the last EDF calibration will be appended to the file and the file name will be appended with -EDF.
General characteristics of using EDF
• The EDF element spreads all points of light within a cellular image into consistent L-shaped patterns. When EDF images are opened in ideas, the data is deconvolved to create an image of the entire cell projected simultaneously in focus.
• During acquisition and before deconvolution, images will appear blurred into characteristic L-shaped patterns and raw max pixel values will be lower with
EDF than with standard mode collection.
• Compensation controls for EDF data can be collected with or without the EDF element in place.
• When analyzing data in IDEAS, after the deconvolution process there will be more light per pixel than in non-deconvolved imagery. Therefore, raw max pixel values may exceed 1023 (for the IS100 instrument) or 4095 (for the ISX).
As long as the images did not saturate the camera during acquisition, these pixel values are valid.
• Object, Morphology and System Masks will be smaller in EDF mode.
• Focus gating is not required. However if there are blurred events due to streaking, these can be removed from the analysis using a focus gate.
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• EDF images exhibit increased texture due to higher resolution. • Brightfield imagery is not as crisp in EDF mode as in standard mode.
• An in-depth discussion of EDF can be found in the following reference:
Cytometry Part A (2007) 71A:215-231
Using MultiMag
The MultiMag option includes 2 additional objective lenses. The 20X lense is useful for very large objects that do not fit into the field of view of the 40X objective such as 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.
To collect a data file using the 20X or 60X object
Objective Field of view Pixel size Depth of field NA
40X 60 um 0.5 um 4 um 0.75
20X 120 um 1 um 8 um 0.5
60X 40 um 0.33 um 2.5 um 0.9
The optional objective can be chosen by selecting the button under Magnification.
When using the 60X obective the core velocity will be reduced to 40 mm/sec instead of the normal 60 mm/sec used during 40X or 20X acquisition. See the section on the
Speed versus Sensitivityfor more information on resolution at different
magnifications.
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Chapter 3
Using the Autosampler
To enable high throughput experiments and unattended operation the autosampler option includes upgraded fluidics, software and an imbedded tray for loading of samples in a 96 well plate format.
Prior to running the plate, a plate definition is created that assigns instrument settings to the wells, names to the output files, and parameters to include in a well plate report that is generated once the plate has completed. While the plate is running, the user may be notified of any errors encountered via email. The instrument can also sterilize at the completion of the plate.
Workflow:
• Create Instrument Setting Template(s) (.ist) to be used for your plate. To do this, run an experimental sample manually with all of the fluorescence dyes to be used in the experiment (see INSPIRE Setup Quick Start Guide). Save each relevant template.
• Create a Well Plate Definition (.def) that assigns instrument settings to wells, names to the sample output files, and parameters to include in the plate report
(see procedure below).
• Add 75 ul samples to the 96 well plate, cover with Sigma-Aldrich X-Pierce
Film (XP-100, Cat # 2722502) and load the plate into the autosampler.
• Run the plate (see procedure below).
Access to AutoSampler operations is found under the AutoSampler menu.
From this menu you may:
• Extend or retract the tray
• Create a plate definition
• Run a plate
• Run a single well from a plate
Define the plate:
The steps required to create a Well Plate Definition (.def) and run a plate are outlined below:
1 Choose Define Plate from the Autosampler menu to open the Well Plate
Definition window.
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Optional upgrades
2 Begin to create a new definition or you may browse for a previously saved definition (to edit) by clicking on the folder icon.
3 Name the plate definition.
4 At a minimum, each well requires an Output File Path, Max Acquisition Time, and
Template File in order to be considered ‘defined’. Other parameters can be added to the definition in the next step.
5 Choose the parameters you would like to use.
• Click Add/Remove Well Parameters to choose the parameters you want to report for the wells.
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There are several categories of parameters that may be chosen as a group or individually. See the list of parameters above. Check or uncheck the desired parameters. The user can also define custom parameters. Expand the category to see the individual parameters. To delete a custom parameter, select it and use the delete key. Click OK when done
• It is highly recommended to leave the validate sample option 'Yes'. See notes below regarding validating samples.
• To include a parameter in the file name, click in the box below the column heading (make sure it says ‘yes’).
• Columns can be re-ordered by click/drag.
• Batching of the data into IDEAS may be done if a compensation matrix and template exist for the experiment.
• Click OK when finished adding or removing parameters.
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Optional upgrades
6 Define the wells. Select wells to define by clicking a) individually (orCtrl click / shift click for multi-select), b) rows or columns, c) color, d) the ‘Select Defined’ button or e) All. In this example column 1 is selected.
Note: Selecting and defining wells with shared parameters first and then refining the definition for sets of wells makes it easier to organize the definition.
7 You can edit values for some of the Custom and many of the Standard parameters. You can do this for all selected wells or for individual wells.
For example, if you want to collect with Max Acquisition Time 10 minutes for the selected wells, type 10 in the 'Apply to selected' box below the Max Acquisition
Time heading. If you want to only apply this to a single well, type this value in the box corresponding to that well.
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.
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• A warning may be displayed if there are undefined or partially defined wells.
Select Yes to return to plate definition or No to continue.
Start the autosampler
10 Click Start to run the plate. The Auto Sampler Unattended Operation window opens with the Plate Definition you just saved.
• If you wish to choose a different Definition, browse for it by clicking 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.
11 Check or uncheck the boxes Return samples, Sterilize, Shutdown.
Note that these boxes may be checked or unchecked while the plate is running and the operation will apply after the current sample is finished.
12 Select the wells to run (they will appear in the list).
13 Click Eject Tray to extend the plate nest.
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During a run:
14 Add at a minimum 75 ul samples to the 96 well plate, cover with Sigma-Aldrich X-
Pierce Film (XP-100, Cat # 2722502) and place your plate on the tray 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) Load, 2) Validation ( flow speed CV, focus, brightfield intensity object rate, 3) Data Acquisition, 4) Result
(success or error).
• You may stop the plate at any time by clicking the Stop button. This does not initiate sterilize (even if the ‘Sterilize after running plate’ box is checked).
• Should the sheath tank or beads reservoir become empty or the waste tank full during a run, an alert will be sent to the email entered in the well plate definition. Acquisition will pause until the user intervenes.
• If an error occurs on a well, the sample is returned, an alert is sent to the email address entered in the well plate definition, and the autosampler moves on to the next well.
• 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)
Report:
17 A well plate report .txt file will be saved (to the folder designated in the Output File
Path of the plate definition) at the end of the run either when it was stopped manually or completed the entire plate.
18 If batching was included in the well plate definition the data files will be processed using the IDEAS compensation matrix and templates designated. All of the .cif , .daf and statistics report .txt files will be saved to the designated output file path.
Notes:
Email notifications:
The parameter 'Error Notification Email' may be selected. Provide the email address for notification. In order for this to work, the system must be enabled as follows.
• From the Instrument menu choose Options.
• Select “Email Notification” tab.
• Your IT department must provide the following informationand add firewall exceptions for INSPIRE and this port.
— SMTP Host
— SMTP Port
— Login
— Password
— To
— From
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Chapter 3
• Cc (optional)
• Choose whether to enable SSL
If the email notifications do not work, have your IT department make sure the messages are not being blocked by firewalls or other network security settings.
Also, try enabling/disabling the SSL check box.
Sample validation:
When using the autosampler, each sample load is validated to ensure good imagery is collected for the sample.
First the core speed and rough synchrony is checked to make sure it is close enough to proceed and this requires Brightfield (if the template has BF off then BF must be turned on). This step occurs whether validation is ON or OFF in the plate definition.
It is highly recommended that the validation is 'Yes' in the plate definition for every well.
When the validation is 'Yes' for the well, the object rate and synchrony is then tested and must pass some predetermined limits. Focus adjustment is enabled. This ensures good imagery is collected.
Finally, if BF was turned ON for the first step then it is turned OFF again for the current template.
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Experimental design
Experimental design
This chapter includes the Sample Preparation Guide which is a guideline to designing the ImageStream experiment.
1 Choice of Cell Type: The particle size should be less than 120um using 20x magnification, 60um using 40x, and 40um using 60x. Images below are THP1 cells (~15um diameter) labeled with FITC NFkB and Draq5.
2 Final Sample Concentration and Volume: At least 1 million cells in 50 m cells/ml) in PBS/2%FBS in a 1.5mL siliconized microcentrifuge tube. Will run
~400 cells per second on low speed.
3 Protocols: In general, any established labeling protocol used for flow cytometry will work with the ImageStream (see Current Protocols in Cytometry for general labeling techniques). Stain cells on ice in the presence of azide when possible to reduce non-specific capping of antibody. Use siliconized polypropylene tubes when possible.
4 Choice of Fluorochromes: Choose fluorochromes that are excited by the lasers in your ImageStream (405,488,642nm are most common). Use the chart on p.3 or look online for a spectra viewer that will help you plan which dyes will work the best.
5 Compensation: Have a sample of cells each labeled with a single-color for each fluorochrome used (i.e. FITC only cells, PE only cells, etc.).
6 Cell Aggregation: Minimize aggregation problems by straining the sample through a 70um nylon mesh strainer, or by using an anti-clumping buffer such as EDTA or
Accumax prior to fixation.
7 Fixation: If fixation is desired, thoroughly fix cells with 1% PFA on ice for 20 min.
8 Number of samples: No more than 30 total for feasibility experiments. Please limit the samples to the following; Positive and Negative biologic controls, compensation controls, and experiment samples.
9 Brightness of Stain and Stain Balancing: Quantifying the location and distribution of signals in an image is a demanding task that requires optimized labeling.
Below are a few suggestions to help design the experiment:
• Try to achieve at least a full log shift in fluorescence, as measured by FACS.
• Use the brightest dye for the antigen with the smallest copy number.
• The brightness of probes can be independently controlled by changing the laser power. However, data quality is enhanced when the brightness levels of all probes excited off a single laser are balanced to within a log of each other.
Probe balancing avoids the saturation of bright stains when they are combined with dim stains in the same sample.
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Chapter 4
Fluorochrome charts
6 channel system
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12 channel system
Experimental design
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Chapter 4: Instrument Calibrations and Tests
Chapter 4: Instrument Calibrations and Tests
ASSIST Tab
ASSIST (Automated Suite of Systemwide ImageStream
X
Tests) is a suite of calibrations and tests for critical subsystems operating within the ImageStream
X
.
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
X
.
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 ASSIST daily to optimize the performance of the ImageStream
X
.
To run ASSIST calibrations and tests:
1 Click Start All Calibrations and Tests to run all standard calibrations and tests.
2 Optional: Click Run Beads to begin running beads without starting calibrations or tests.
3 To run 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.
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Reporting ASSIST data
ASSIST calibration and test results are stored in a database that can be reported.
To view the last result of a specific calibration or test click on the button to the right of that item or click on the History button and choose the routine and date you wish to view.
To create a report for all calibrations and tests click on the Generate Report button. A window opens that allows you to choose the dates for the report.
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Chapter 4: Instrument Calibrations and Tests
ASSIST Calibrations
The calibrations in the current suite are described in detail below.
Camera Synchronization Calibration
Measures and stores a magnification calibration (camera synch) factor relating the
Flow Speed Detection frequency and the camera clock rate. This factor is used to maintain synchronization between the moving imagery projected onto the camera surface and the electronic charge resulting from that imagery. Proper synchronization helps ensure crisp image collection.
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.
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Chapter 5
Spatial Offsets Calibration
Measures and stores 12 calibration factors for the vertical and horizontal registration of each spectral channel of the ImageStream
X
. Many assays that are run on the
ImageStream
X 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
X must maintain subpixel spatial registry between channels.
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, if the 12 channel option is present, this calibration will illuminate and calibrate all 12 channels.
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,
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Chapter 4: Instrument Calibrations and Tests 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
X 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.
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 installed the Dark Current calibration will be simultaneously performed for both cameras.
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
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Chapter 5 remaining five channels. The purpose of this calibration is two fold. First, the spectral leakage values are used to spectrally correct the imagery in IDEAS by removing any Brightfield light leakage from the other five channels. The second purpose is to ensure that the spectral characteristics of the instrument remain constant over time. The Brightfield cross talk calibration will simultaneously calibrate leakage from all eleven channels if the 12 channel option is installed in the instrument.
Core Stage Position Calibration
The alignment of the stage in the X direction is controlled so that the position of the core is centered in the field. This calibration finds the core position using the X centroid position of the SpeedBeads and calculates an offset from the factory setting and sets the position of the stage in the X dimension.
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Chapter 4: Instrument Calibrations and Tests
Horizontal Laser Calibrations
The alignment of each laser in the ImageStream
X is automatically controlled to ensure optimal performance via the Horizontal Laser Calibration. The calibration routine sweeps the horizontal position of the laser across the flow stream. At each of
15 predefined intervals during the sweep, 1000 SpeedBead images are collected and analyzed to determine the intensity of each bead. The median intensity for each position is then plotted and fit to a fourth order polynomial. The peak height of the polynomial is then determined. This position is the point where the peak intensity of the Gaussian laser beam intersects the center of the flow core. This position provides both the highest intensity for illuminating the core stream and the point with the lowest coefficient of variation. This position is stored for each laser and used as the default position during subsequent assays.
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The result for the calibration are shown below the list when the calibration is selected.
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.
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Chapter 4: Instrument Calibrations and Tests
Retro Calibration
The ImageStream
X 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.
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Chapter 5
Autosampler Nest Calibration
The ImageStream
X autosampler runs a self calibration. This calibration verifies that the sipper can self-calibrate and find the home position. If the calibration fails or is not run the autosampler will not run.
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Chapter 4: Instrument Calibrations and Tests
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.
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Chapter 5
The results and limits of the test are shown below the list when the test is selected.
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Chapter 4: Instrument Calibrations and Tests
BF Intensity Selection Test
Verifies the BF intensity calibration for each BF mode. The image intensity must reach 200 within 20 iterations. If this test fails, the user should run the BF Intensity
Selection Calibration individually and then re-run the test.
The results and limits of the test are shown below of the list when the test is selected.
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Chapter 5
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). Nonuniformities 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.
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Chapter 4: Instrument Calibrations and Tests
Camera Noise Test
The electronic noise is measured with no illumination to the CCD in two successive frames. The fluctuation is measured on a pixel by pixel basis.
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Chapter 5
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
X 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
X 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.
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 are stored in the ASSIST database. The results and limits of the test are shown below the list when the test is selected.
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Chapter 4: Instrument Calibrations and Tests
Flow Core Lateral Stability Test
Provides a statistical characterization of the stability of the core in the direction lateral to flow. The test computes the centroid position of approximately 3000
SpeedBeads. During the test a histogram of bead centroid position is plotted in the test window. When the test is complete, the standard deviation of bead centroid position (in pixels) is printed in the test window.
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.
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Chapter 5
Flow Core Position Test
Measures the position of the core relative to its ideal position within the flow cuvette.
The ImageStream
X uses sheath flow to hydrodynamically focus objects within a precise region in the cuvette. Improper sheath solution, protein buildup, microbubbles and other factors can alter the position of the core within the cuvette. If this occurs, the photometric and morphological measurement repeatability may degrade.
This test measures the current core position and compares it to the ideal location of the core as determined in the manufacturing process. The deviation from the ideal position is reported in microns and stored in the ASSIST database.
The result and the limits for the calibration are shown below the list when the calibration is selected.
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Chapter 4: Instrument Calibrations and Tests
Focus Offset Beads Test
Measures the offset between the focus determined by the AFFS system and location of the peak response of the Image Collection system. This test performs a pan through focus while simultaneously collecting SpeedBead focus data from the
AFFS system and SpeedBead image data from the image collection system. The
AFFS data are processed to find the zero crossing (point of no defocus) and the image data are processed to determine the peak response (point of highest spatial resolution). Both sets of data are plotted as a function of Z position along the horizontal axis. The AFFS zero crossing and image collection system peak response are indicated vertical lines and numerical results are reported to the Focus
Offset test tab. The difference (in microns) between these two positions is determined and compared against predetermined limits and stored in the ASSIST database. If the MultiMag option is installed, a focus offset test will be performed for each magnification.
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Chapter 5
Focus Percentage Test
Measures the percentage of SpeedBeads in focus and sets a limit of 90%.
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Chapter 4: Instrument Calibrations and Tests
Focus Uniformity Test
Measures the best focus position for every channel and then calculates the difference of each channel from the mean for all channels. The tolerance for focus uniformity ensures that all channels are in optimal focus.
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Chapter 5
Image Quality Ensquared Energy Test
Measures the ability of the optical system to resolve fine details in the image using ensquared energy ratio. The optics term ensquared energy refers to a measure of concentration of energy in an optical image when quantifying image sharpness for digital imaging cameras using pixels. The ensquared energy ratio is one of several parameters often used in the design of high resolution optical systems to characterize their performance. In this ASSIST test, the ensquared energy ratio of a
3x3 pixel array centered within an 11x11 pixel array is determined and compared against predetermined limits. The test is designed to measure the optical quality of the image independent of focus, lateral core stability, and axial core stability. During the test approximately 5000 SpeedBead images are collected over a range of focus positions. The imagery is analyzed during collection by computing the ensquared energy ratio in each image. The ensquared energy for each image at each focus location is shown in a plot. The mean ensquared energy for each focus position is noted as a dark blue data point for each focus position. The ensquared energy for the 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.
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Chapter 4: Instrument Calibrations and Tests
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Chapter 5
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.
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Chapter 5: Troubleshooting
Chapter 5: Troubleshooting
This section is designed to help you troubleshoot the operation of the
ImageStreamX-Mark II. If additional assistance is required, contact the Amnis service department.
System
•
Unstable fluidics (Air or clog in system)
•
•
• Event rate is slower than expected
•
Cross-contamination from previous samples
•
Erroneous fluid level indicator
•
Instrument will not pass ASSIST
•
Compensation wizard fails to complete
Software
•
•
•
Plots fail to update, or update slowly
• Data file fails to collect
Image
•
•
Imaging and acquisition rate is erratic, or appears frozen
•
•
Objects are not centered in the channel
•
Objects are rotating in the core stream
•
Objects are out of focus or distorted
•
•
The two brightfield images are not of the same cell
•
Images appear pixelated or larger than normal
•
Objects appear larger or smaller than normal
•
Not all 12 channels are being displayed
• Images have incorrect colors
Intensity
•
Fluorescence imagery appears too dim
•
Fluorescence is too bright, images have a contrasting color or appear flat
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Chapter 6
•
One channel saturates while the others do not
•
Scatter is too dim or bright or changes over time
•
Large variation in brightfield intensity levels
•
Brightfield intensity level sets incorrectly
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Chapter 5: Troubleshooting
Symptom Possible Causes
Unstable fluidics
Air bubbles in the
(Air or clog in system)
sample
Air bubbles in fluid lines
Clog in fluid lines
Sample is too concentrated
Inappropriate sheath solution
Fluid lines are leaking
SpeedBeads fail to run
System
Recommended Solutions
Make sure a sufficient sample volume is used. To clear the air bubble: Run the purge bubbles script.
Detergents and foaming agents (such as
FBS) can cause bubbles to form in the lines. If these buffers are causing air in the system remove them from the sample and resuspend in dPBS. Run the purge bubbles script.
Run the sterilize script, followed by the startup script. Load calibration beads and verify the system runs normally.
Filter the sample with a 70um nylon cell strainer. Run the sterilize script, followed by the startup script. Load calibration beads and verify the system runs normally.
Clumpy and viscous samples cause cavitation in the fluidic lines and create bubbles. Dilute the sample to 1x10^7 cells/ml and strain the cells through a
70um nylon mesh. Run the purge bubbles script.
Verify the sheath is dPBS. De-gas the sheath as appropriate. Third party sheath buffers cannot be used.
With the system powered down look for leaking sheath. Verify the fluid lines 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 histograms have tight CVs at the appropriate core velocity. To view bead images select the All population and check include beads.
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Chapter 6
Symptom Possible Causes Recommended Solutions
Fluidics respond sluggishly
Event rate slows over time
Event rate is slower than expected
Air buffer in the sheath syringe is not correct
Fluid lines are leaking
Cells have settled in the lines
The sheath syringe should contain 2-4 ml of air to buffer the movement of the pump’s microstepper motor. If too little air is present run the “start-up” script.
With the system powered down look for leaking sheath. Verify the fluid lines mount snuggly into position. Call Amnis service.
Cells settle in the lines after 45-60 minutes 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.
Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics .
There is a clog or air bubble in the system
Sample syringe is empty
Sheath syringe is empty
Fluid lines are leaking
Load a fresh sample
Load sheath, then go to the instrument drop down and run prime.
With the system powered down look for leaking sheath. Verify the fluid lines mount snuggly into position. Call Amnis service.
Sample concentration is low
Make sure the sample concentration is between 10
7 and 10
8 cells/ml. Lower concentrations can be used but this will decrease the cells/second.
Core is off center
Insufficient
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 dropdown menu.
See solutions for unstable fluidics.
Turn the appropriate lasers on. Set the
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Chapter 5: Troubleshooting
Symptom
Crosscontamination from previous samples
Erroneous fluid level indicator
Possible Causes Recommended Solutions
illumination
Cells are not displayed due to over clipping.
DNA dye from previous sample is labeling current sample
Cells from the previous sample are appearing in current sample
Tank has moved away from the sensor
Sensor is broken
785 SSC laser to 40mw. Set the laser powers to maximum and decrease them to prevent pixel saturation.
For large diameter cells go to the advanced drop down, select acquisition and check the box labeled keep clipped objects.
DNA dyes must be thoroughly flushed from the sample lines, to prevent residual dye from labeling subsequent samples.
Load a sample of 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 sample of 10% bleach followed by a PBS wash to remove most contaminating cells, or run the sterilize script (~30min).
Open the buffer compartment and move the tank closer to the sensor until the fluid level indicator is correct.
Power down and power up the instrument, if this does not fix the problem, call Amnis service.
Instrument will not pass
ASSIST
Incorrect template loaded
Go to the file drop down and select “load default template”. Re-run ASSIST.
SpeedBeads fail to run
Verify the beads will run by returning any sample, going to fluidics section and press stop, then run. Next go to the advanced drop down, select flow speed, and check that the red and black histograms have tight CVs at the appropriate core velocity. To view bead images select the All population and check include beads.
SpeedBeads are not running properly
The particles must be running >1000 events per second, and without significant clumping. If the beads are diluted or clumped, try running a fresh
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Chapter 6
Symptom Possible Causes Recommended Solutions
Calibration and/or test failure tube of beads. If the problem persists there may 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. Rerun the test by clicking in the box next to the test, and pressing the start button in the popup window. If the test fails three times in a row, call Amnis service.
Focus adjustor calibration failure
Frame Offset calibration failure
Spatial Offsets calibration failure
Verify brightfield is working properly.
Verify brightfield is working properly.
Verify brightfield is working properly.
Dark Current calibration failure
Brightfield XTalk calibration failure
Horizontal Laser
Calibration failure
Retro Calibration failure
Side Scatter
Calibration failure
Laser Power test failure
Make sure the excitation lasers are off and brightfield is blocked. Completely power down the instrument and power back up to re-run the test.
Verify brightfield is working properly, and that spatial offsets passed.
Verify the laser turns on and can set power properly. Completely power down the instrument and power back up to rerun the test. Verify spatial offsets passed.
Verify the laser turns on and can set power properly. Verify spatial offsets and frame offsets passed.
Verify the 785 SSC laser turns on and can set power properly. Completely power down the instrument and power back up to re-run the test. Verify spatial offsets passed.
Verify the laser turns on and can set power properly. Completely power down the instrument and power back up to rerun the test. Verify spatial offsets and frame offsets passed.
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Chapter 5: Troubleshooting
Symptom
Compensation wizard fails to complete
Possible Causes Recommended Solutions
Brightfield alignment test failure
Brightfield uniformity test failure
Verify brightfield is working properly.
Verify brightfield is working properly.
Camera noise test failure
Flow Core Axial
Stability test failure
Flow Core Lateral
Stability test failure
Flow Core Position test failure
Focus Percentage test failure
Focus Uniformity test failure
Image Quality test failure
The region to collect was set incorrectly
Verify camera can image properly.
Completely power down the instrument and power back up to re-run the test.
Verify the reagent buffers are full. Run the sterilize script followed by the startup script, and re-run the test. See solutions for unstable fluidics.
See solution above ( Flow core Axial stability test ).
See solution above ( Flow core Axial stability test ).
See solution above ( Flow core Axial stability test ).
See solution above ( Flow core Axial stability test ).
See solution above ( Flow core Axial stability test ).
In the wizard verify that 1,000 of “All” cells (or of a region drawn on the appropriate population) are being collected.
Too many objects are being collected
Set the events to acquire less than 1,000.
Cells are not fluorescent
Cells are stained with more than one fluorochrome
Make sure that the compensation control sample has more than 10% positive events, and are as bright as possible. IgG capture beads or a cell line stained with a single fluorochrome may be used for comp controls.
Compensation controls must be a sample with a single fluorochrome label in a single tube. Each fluorochrome must be run separately.
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Chapter 6
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Chapter 5: Troubleshooting
Symptom
INSPIRE appears to freeze
INSPIRE fails to launch
Software
Possible Causes Recommended Solutions
Camera is not running
If the camera is already running
A script is running
The INSPIRE application has crashed
Click Run/Setup.
Click Stop then Run/Setup
Imaging is paused Click Resume.
No objects in the current image view mode
In the cell view area, select the all population.
Wait until the script completes, or if necessary, click Abort Script to prematurely stop the operation.
Open the Windows Task Manager by pressing <Ctrl + Alt + Del>. Click the
Applications tab. If INSPIRE is ‘Not
Responding’, select the INSPIRE task and click End Now. Restart the
INSPIRE application by double clicking the icon on the desktop. If the program restarts, make sure the lasers and brightfield lamp are turned on and then re-establish the core stream. If the application does not start, use the
Windows Task Manager to end the
INSPIRE task again. Shut the instrument and computer down from the
Start menu. Then turn on the instrument as described. If a crash occurs during the day, a complete shutdown is recommended at the end of the day, before running sterilize.
Splash screen is not responding
Loss of communication between the computers and instrument.
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.
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Chapter 6
Symptom Possible Causes Recommended Solutions
Plots fail to update, or update slowly
Data file fails to collect
Computer resources are being over used
Close all third party software.
Too many plots in the template
For optimal plot update rates limit the number of plots to 15.
Parent population has no qualifying events
Plots are scaled incorrectly
Right click on the plot, select graph properties, and change the selected population to “all” or a population that has qualifying events.
In the plot tool bar, press the – magnifying glass and rescale the plot.
No events qualify for the region
Computer hard drive is full
Data file collected rapidly
File directory was lost
No .rif or .fcs file was created
Make sure there are events going into the collection region by viewing that region in the image gallery and updating the acquisition collection population appropriately.
Verify the cell concentration is appropriate. 1x10^7 cells/ml is ideal.
Verify the computer hard drive has sufficient 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 displayed. Backup and delete data to free up disk space.
Some samples have high concentrations and acquire faster than the display rate. Check the destination folder and see if the raw data was collected.
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 destination folder is accessible using the browse button in the Acquisition
Settings section.
Go to the file drop down menu and check Generate .rif and or .fcs file.
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Chapter 5: Troubleshooting
Chapter 6
Symptom
No Images
Imaging and acquisition rate is erratic, or appears frozen
Image
Possible Causes Recommended Solutions
Camera is not running
If the camera is already running
Insufficient illumination
Click Run/Setup.
Click Stop to stop the camera, and then click Run/Setup.
Imaging is paused Click Resume.
Displayed region is incorrect
In the cell view area, select the all population.
Turn the appropriate lasers on. Set the
785 SSC laser to 40mw. Set the laser 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 outside the objective’s field of view
Manually find the core stream. In the focus and centering section, move core track left or right to find the core.
Computer resources
Close all third party software.
are being over used
Sample concentration is low
Make sure the sample concentration is between 10
7 and 10
8 cells/ml. Lower concentrations can be used but this will decrease the cells/second.
Region being viewed has few or no cells
Insufficient illumination
In the cell view area, select the all population, or readjust regions to include more cells.
Turn the appropriate lasers on. Set the
785 SSC laser to 40mw. Set the laser powers to maximum and decrease them to prevent pixel saturation.
Make sure the brightfield lamp is turned on and click Set Intensity.
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Chapter 5: Troubleshooting
Symptom
Objects appear streaked
Objects are not centered in the channel
Possible Causes Recommended Solutions
The sample is too concentrated
The process of object detection can safely handle up to 4000 objects per second. The maximum sample concentration is 4-5x10^8 cells per ml, with the recommended concentration
1-10 x10^7 cells per ml. To decrease the event rate, dilute the sample.
The sample has an excessive amount of debris
Use a region to eliminate the debris from the data file, or prepare a fresh sample.
Computer resources
Exit all third party programs.
are being overused
Camera is not tracking the cell velocity accurately
SpeedBeads fail to run
There is only one channel of brightfield
Verify brightfield is working normally and rerun ASSIST using calibration beads.
See solutions for unstable fluidics.
Verify the beads will run by returning any sample, going to fluidics section and press stop, then run. Next go to the advanced drop down, select flow speed, and check that the red and black histograms have tight CVs at the appropriate core velocity. To view bead images select the All population and check include beads.
Verify that there are two brightfield channels. Check in the image display properties that 1 and 9 are active, verify that brightfield is emitting in channels 1 and 9.
Lateral deviation of the core stream due to air or clog in the system
Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Autofocus and centering is not tracking properly
In the Focus and Centering section, adjust focus and centering left or right, until the images are centered and in optimal focus.
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Chapter 6
Symptom Possible Causes Recommended Solutions
Objects are rotating in the core stream
Objects are out of focus
Core stream position is grossly off-center within the flow cell due to air or clog in the fluidics
Autofocus is not tracking properly
The core tracking and focus tracking should not change significantly from day to day. If either value changes radically, objects may rotate due to interactions with the sheath. An offcenter core stream is caused by air or clogs in the fluidic system. Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Camera line rate is incorrect
Re-run the focus adjuster and frame offset calibration in ASSIST, and verify it passes.
Excessive core stream variation due
Run the purge bubbles script from the to air or clog in the fluidics instrument drop-down menu.
See solutions for unstable fluidics
.
Core stream is moving too fast for the camera
Allow the system to settle for 60 seconds after loading a sample.
Collect data once imagery looks good.
In the Focus and Centering section, adjust focus and centering, left or right, until the images are centered and in optimal focus.
Data is binned
EDF is on
Turn off bin mode by selecting fluidics, and set the slider bar to low speed high sensitivity. Verify this change in the advanced drop down by selecting camera and verifying a 1x bin mode.
In the magnification and EDF section uncheck the EDF checkbox.
Objects are cropped
Lateral deviation of the core stream due to air or clog in the system
Run the purge bubbles script from the
instrument drop-down menu . See solutions for unstable fluidics.
Autofocus and centering is not tracking properly
Incorrect
In the Focus and Centering section, adjust focus and centering left or right, until the images are centered and in optimal focus.
In the magnification and EDF section
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Chapter 5: Troubleshooting
Symptom Possible Causes Recommended Solutions
The two brightfield images are not of the same cell
Images have incorrect colors
Magnification
Frame offset is incorrect
Illumination is grossly misaligned
Cross correlation is incorrect
Channel is not activated
Image gallery display is set up incorrectly 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 calibration routine, and verify it passes.
Call service, and verify that the illumination pathways are in proper alignment.
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.
Images appear pixelated or larger than normal
Image gallery zoom is active
Objects appear larger or smaller then normal
Incorrect magnification
Not all 12 channels are being displayed
Image gallery zoom is active
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 channel color for the channel.
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Chapter 6
Symptom
Intensity
Possible Causes Recommended Solutions
Fluorescence imagery appears too dim
Fluorescence is too bright, images have a contrasting color or appear flat
Image display settings are set too low
Sample did not label well
Insufficient illumination
Excitation laser is misaligned
Image display settings are set too high
Instrument sensitivity is set too high
Increase the image display gain by clicking on the channel column, selecting the appropriate channel, and moving the right green handle bar to a smaller value, or the brightest pixel in the histogram. To set the display background to black move the left green handle bar to the dimmest pixel in the histogram.
Look at the sample with a fluorescent microscope.
Turn the appropriate lasers on. Set the laser powers to maximum and decrease them to prevent pixel saturation.
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.
Core stream position is grossly off-center within the flow cell due to air or clog in the fluidics
Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Run calibration particles on the Flow
Sight. Load the default template. Open
ASSIST, re-run the laser alignment calibration for the appropriate laser line, and verify it passes.
Decrease the image display gain by moving the right hand green handle bar to a higher value for the appropriate camera channel.
Decrease the excitation laser power to prevent pixel saturation. Saturation is indicated in the image gallery by pixels colored in a contrasting color
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Chapter 5: Troubleshooting
Symptom
One channel saturates while the others do not
Scatter is too dim or bright or changes over time
Possible Causes Recommended Solutions
The sheath syringe is empty
There is a clog or air bubble in the system
(generally red or white).
Set the brightfield intensity to 800 counts by pressing “Set Intensity”.
Load sheath, then go to the instrument drop down and run prime.
Run the Purge Bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Instrument sensitivity is not optimized
Probing protocol requires better stain balance
Excessive fluorescent dye is left in the sample buffer.
The best instrument setup maximizes the dynamic range of fluorescence signal, while at the same time avoiding image pixel saturation
(which cannot be compensated). In general decreasing the laser powers until no pixels saturate.
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, however if too much dye is in solution it can cause the core stream to fluoresce. It’s important to balance the concentration of these dyes so that the cells can be imaged properly.
Typically the concentrations in
“Current Protocols in Cytometry” should work.
Instrument is experiencing large temperature variation
Allow the instrument to warm up by running for 15 minutes.
Laser power set too high or low
Direct a fan toward the back of the instrument to dissipate excess heat, or move the system to a temperature controlled environment.
Increase or decrease the 785 SSC laser power.
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Chapter 6
Symptom Possible Causes Recommended Solutions
Core stream position is grossly off-center within the flow cell due to air or clog in the fluidics
Large variation in brightfield intensity levels
Large flow speed variation due to air
Light source delivering variable output
Brightfield intensity level sets
Intensity set before desired flow speed
incorrectly
has been achieved
Movable optics are out of position
Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Run the purge bubbles script from the instrument drop-down menu.
See solutions for unstable fluidics.
Power down and power up the instrument, 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 instrument, if this does not fix the problem, call Amnis service.
- 94 -
Index
Acquisition
Brightfield crosstalk coefficient 57
flow core lateral stability 69
A
- 95 -
Index
Chapter 7
image quality ensquared energy test 74
B
Brightfield
Brightfield Crosstalk Coefficient 57
Calibrations and Tests
Centering
C
- 96 -
Compensation
Debris
DNA dye
EDF
Features
D
E
F
- 97 -
Index
Chapter 7
Focus
Graphs
histogram 19 refresh 19 scatterplot 19
G
I
ImageStream
- 98 -
INSPIRE
Laser
Magnification
Mask
Menus
L
M
O
- 99 -
Index
Chapter 7
Sample
Saturation
sheath bottle 14 sheath fluid 14
SpeadBeads
Speed
P
R
S
- 100 -
SpeedBeads
Test
Flow Core Lateral Stability 69
T
U
- 101 -
Index
Chapter 7
W
- 102 -
advertisement
Key Features
- Multispectral imaging flow cytometry
- Acquisition of up to 12 channels of cellular imagery
- Large numbers of digital images per sample
- Numerical representation of image-based features
- Statistical significance afforded by large sample sizes
- Combines information content provided by standard microscopy
- Provides information comparable to standard flow cytometry
