Takara Bio SMARTer ICELL8 cx User manual

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Takara Bio USA, Inc.
ICELL8® Imaging
System User Manual
Cat. No. 640000
(071919)

ICELL8® Imaging System User Manual
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Table of Contents
I. Preface............................................................................................................................................................................. 3
II. Introduction..................................................................................................................................................................... 4
III. Startup......................................................................................................................................................................... 7
A. Starting the Hardware ................................................................................................................................................. 7
B. Starting the Software................................................................................................................................................... 8
IV. Operation..................................................................................................................................................................... 9
A. Setting Up the Chip..................................................................................................................................................... 9
B. Focusing on Cells...................................................................................................................................................... 12
C. Acquiring Images...................................................................................................................................................... 17
D. System Shutdown and Next Steps ............................................................................................................................ 17
Appendix A: Maintenance and Troubleshooting .................................................................................................................. 18
Table of Figures
Figure 1. Software interface for cell imaging. ........................................................................................................................ 4
Figure 2. Workflow diagram depicting single-cell isolation and imaging steps..................................................................... 5
Figure 3. ICELL8 Imaging System Components.................................................................................................................... 6
Figure 4. Starting the Mercury Burner.................................................................................................................................... 7
Figure 5. Starting the Camera. ................................................................................................................................................ 7
Figure 6. Turning on the Filter Controller and Stage Controller. ........................................................................................... 7
Figure 7. Starting the Micro-Manager software: Copyright window and Main window........................................................ 8
Figure 8. Opening the Microscope shutter.............................................................................................................................. 8
Figure 9. Main window and Live window.............................................................................................................................. 9
Figure 10. Main window and Multi-Dimensional Acquisition window. ................................................................................ 9
Figure 11. Live window with image of 6 x 6 well region..................................................................................................... 10
Figure 12. Multi-Dimensional Acquisition window. ............................................................................................................ 11
Figure 13. Seating a chip on the microscope holding platform. ........................................................................................... 11
Figure 14. Positioning the ocular tube over the chip. ........................................................................................................... 12
Figure 15. Moving the Motorized Stage to Position 0.......................................................................................................... 12
Figure 16. Setting the illumination channel to DAPI............................................................................................................ 12
Figure 17. Adjusting camera depth. ...................................................................................................................................... 13
Figure 18. Adjusting contrast................................................................................................................................................ 13
Figure 19. Adjusting microscope focus. ............................................................................................................................... 14
Figure 20. Finding a focal point for single cells. .................................................................................................................. 14
Figure 21. Moving the Motorized Stage to Position 143. ..................................................................................................... 15
Figure 22. Setting the illumination channel to Texas Red. ................................................................................................... 15
Figure 23. Brightly lit nanowells containing fiducials.......................................................................................................... 15
Figure 24. Checking cell-stain channels. .............................................................................................................................. 16
Figure 25. Entering chip number and directory location for images. ...................................................................................16
Figure 26. Acquisition of field of view images with DAPI and Texas Red filters. .............................................................. 17
Figure 27. Chip Holder. ........................................................................................................................................................ 17

ICELL8® Imaging System User Manual
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I. Preface
About this Manual
This manual provides instructions for safe and successful operation of the imaging system included with the
ICELL8 Single-Cell System (Cat. No. 640000). Please be sure to review the information in this manual
thoroughly before using the equipment.
For detailed information about performing image analysis following image acquisition, please refer to the
CellSelect® Software User Manual.
Symbols and conventions
The following symbols and conventions are used throughout this manual.
Symbol
Description
WARNING: Indicates a potentially hazardous situation that could result in injury to
the user or damage to or destruction of the system.
CAUTION: Indicates a hazard that could result in loss of data or damage to the
system.
Indicates the presence of an electrical shock hazard and to proceed with caution.
This symbol may appear next to either a WARNING or a CAUTION.
Indicates the presence of a biological hazard and to proceed with caution. This
symbol may appear next to either a WARNING or a CAUTION.
NOTE: NOTE: Provides helpful ancillary information to support the use of the system.
Safety Information
Consult user manuals for corresponding ICELL8 Single-Cell System components for specific safety information.
Operating conditions:
•Operate the imaging system inside an appropriate building. Do not operate the imaging system outside or
in wet environments.
•Block any direct, high-intensity light such as direct sunlight, which may affect the uniformity of the
collected images.
Instrument use:
WARNING: Use of equipment and reagents for cell preparation and isolation with
the ICELL8 Single-Cell System may cause exposure to toxic or biohazardous
chemicals, thereby presenting a hazard. Always wear appropriate personal protective
equipment (PPE), which should at minimum include gloves, eye protection, and a
lab coat, when handling equipment and reagents and operating the imaging system.
CAUTION: Do not position the equipment so that it is difficult to operate the power
switches or remove the power cords.

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WARNING: Use only the power cords provided by the manufacturers. Do not
replace the power cords with inadequately rated cords.
Moving and lifting the system:
WARNING: If you need to move system components after the system has been
installed, ensure that all components are off. Use proper lifting techniques and
appropriate moving equipment.
Warning labels on the instrument:
Note and heed all warning labels on the system components.
II. Introduction
Welcome to the ICELL8 Imaging System
The ICELL8 Imaging System consists of a precision microscope, camera, high-intensity light source, automated
stage, and color filter control for scanning and acquiring images of live single-cell specimens dispensed into an
ICELL8 chip. The Micro-Manager software takes 288 images through DAPI and Texas Red filters from 144
fields of view (6 x 6 wells in each field of view) in approximately 7 minutes. The cell specimens can be quickly
returned to cold storage while the images are analyzed.
Figure 1. Software interface for cell imaging.

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Workflow Diagram
The imaging system described in this user manual is provided as a component of the ICELL8 Single-Cell System.
For detailed information about other ICELL8 components, as well as compatible kits and reagents, please refer to
corresponding documents. The schematic below depicts the role of the imaging system within a typical workflow
for single-cell analysis.
Figure 2. Workflow diagram depicting single-cell isolation and imaging steps.

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System Description
Figure 3. ICELL8 Imaging System components.
Micro-Manager software: Micro-Manager is open source software, created by the Vale Laboratory at UCSF and adapted
for use with the ICELL8 Imaging System. Micro-Manager software controls the motorized stage, optical filters, and
camera. It also acquires and manages microscope images.
Camera: The CMOS digital camera is mounted over the microscope and creates 4-megapixel images at 30
frames/second.
Barcode Reader: The hand-held Barcode Reader is connected to the computer and is used to input chip ID information
by reading the barcode on the back of the ICELL8 chip.
Motorized Stage, Stage Controller, and Joystick: These components function together to facilitate both manual and
automated stage movement. Automated stage movement occurs in synchronization with the Micro-Manager software.
Microscope: As the core component of the imaging system, the microscope provides 4X magnification and fine-tune
focusing, and is specifically optimized for the ICELL8 system.
Filter Controller, Emission Filters, and Filter Wheel: The Lambda Filter Controller automates filter changing during
scanning and image acquisition. The Emission Filters are housed in the Filter Wheel, which is mounted below the
Camera.
Mercury Burner and Power Supply: The Mercury Burner provides high-intensity light to view and acquire images of
sample cells.

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III. Startup
A. Starting the Hardware
1. Turn on the Mercury Burner Power Supply (Figure 4, below) and allow it to warm up for at least 5
minutes. It will emit faint clicks before turning green. When the light is solid green, it is ready.
NOTE: If the light does not turn on and the faint clicks stop, turn the switch off and wait at least 5
minutes before turning it on again.
Figure 4. Starting the Mercury Burner.
CAUTION: The Mercury Burner is a high-intensity light source; handle carefully.
•After the burner is turned on, allow it to run for 30 minutes before turning it off.
•Wait for at least 30 minutes before turning on a previously turned-off burner.
•Do not turn the burner on and off more than 4 times in a day; it should be left on
if you plan to use it frequently throughout the day.
2. Turn on the Camera and allow it to start up until the light is solid green. If you start the Micro-
Manager software before the camera has completed the startup you will receive an error. If the error
occurs, close Micro-Manager and restart the camera.
Figure 5. Starting the Camera.
3. Turn on both the Filter Controller and the Stage Controller.
Figure 6. Turning on the Filter Controller and Stage Controller.

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B. Starting the Software
1. Turn on the computer and launch the Micro-Manager software:
NOTE: Wait until all hardware is on and ready before starting the software, otherwise the software
will generate an error message.
2. Once the Micro-Manager Copyright window opens, click OK to continue to the Main window.
Figure 7. Starting the Micro-Manager software: Copyright window and Main window.
3. Ensure that the manual shutter is in the “Open” position.
Figure 8. Opening the Microscope shutter.

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IV. Operation
A. Setting Up the ICELL8 Chip
1. Click Live in the Main window. The Live window (right) opens and displays a continuous feed from
the single ocular tube of the microscope.
Figure 9. Main window and Live window.
2. Click Multi-D Acq. to open the Multi-Dimensional Acquisition window (right).
Figure 10. Main window and Multi-Dimensional Acquisition window. The Multi-Dimensional Acquisition window
acquires stacks of multiple images using various parameters. For single-cell images of ICELL8 chip nanowells, the
software uses the “Multiple positions (XY)” setting to acquire images of 6 x 6 well regions.

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NOTE: The wells will appear blurry as you focus below the top of the well to find the cells near the
bottom of the well.
Figure 11. Live window with image of 6 x 6 well region.
3. Make sure that the Multiple positions (XY) box is checked (see Figure 12, below).
4. Click Load Chip type and load the configuration file 72.72.pos (if it is not preloaded).
5. Position the cursor so that it resides in the Chip Number field (see arrow in Figure 12, below).
6. Input the chip number, or use the Barcode Reader to scan the two-dimensional barcode on the back of
the chip.
CAUTION:Be sure to avert your eyes from the scanner light emitted by the
Barcode Reader.

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Figure 12. Multi-Dimensional Acquisition window.
7. Place the chip on the microscope Holding Platform with the chamfered (notched) corner facing the
upper right corner (see Figure 13, below). Ensure that the chip is sitting flat on the chuck from all
sides and is in the correct orientation.
NOTE: The ship should be centrifuged prior to imaging.
Figure 13. Seating a chip on the microscope holding platform.

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8. Remove the second release liner. Hold down the tab of the Imaging Film while peeling off the top
layer release liner of the Imaging Film using tweezers. Save the peeled release liner. The chip will
remain sealed by the double-sided intermediate film layer.
9. Move the ocular tube over the chip (Figure 14, below). Ensure that the ocular tube is vertical and
centered (you should hear/feel a slight click).
Figure 14. Positioning the ocular tube over the chip.
B. Focusing on Cells
1. Select the row Pos0 in the Stage Position List window (Figure 15, below), and then click Go to.
Figure 15. Moving the Motorized Stage to Position 0.
2. In the Main window, under “Configuration settings”, select DAPI as the illumination channel for
examination of Hoechst-stained cells.
Figure 16. Setting the illumination channel to DAPI.

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3. In the lower portion of the Main window, select the Contrast tab and select a value in the range of
12–14 bits in the Camera Depth menu.
Figure 17. Adjusting camera depth.
4. Adjust the contrast of the chip image by selecting Autostretch and Ignore and a value in the range of
1–3% for Ignore (see Figure 18, below). If this does not provide optimal contrast, drag the ends of
the diagonal line (see arrows in Figure 18, below) closer to the histogram peaks and zoom out when
satisfied with the focus in the Z-plane.
Figure 18. Adjusting contrast.

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5. Use the microscope manual focus (under the stage on both sides; see Figure 19, below) to achieve the
optimal Z-plane focal depth to visualize a field of single cells.
Figure 19. Adjusting microscope focus.
6. Use the zoom function in the tool bar (indicated by blue box in screenshot below) for close-ups. Once
selected, use +/- keys or left/right mouse buttons to zoom in and out.
•Select a cell or couple of cells that are off center. Adjust the fine adjustment knob back and
forth to find the focal point of a cell (smallest, sharpest view), or focal point between two cell
depths (see Figure 20, below).
Figure 20. Finding a focal point for single cells.
•Select Pos143 and click Go to (see Figure 21, below). Check the cells at Position 143 to
ensure that most of the cells have good resolution.
NOTE: If the cells are truly out of focus, ensure that the chip is perfectly flat on the Dispense
Platform.

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Figure 21. Moving the Motorized Stage to Position 143.
7. In the Main window, under “Configuration settings”, select Texas Red as the illumination channel
for examination of fiducial-containing wells.
Figure 22. Setting the illumination channel to Texas Red.
8. Confirm that five fiducial-containing wells are present, forming an “X” shape at Pos0 (Figure 23, below).
There are four fields of view (Pos0, Pos11, Pos132, Pos143) across an entire chip that include
fiducial-containing nanowells. Each field of view includes 36 nanowells (a 6 x 6 grid).
Figure 23. Brightly lit nanowells containing fiducials.

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9. Select Pos143 in the Stage Position List window and confirm that all 36 nanowells are properly
captured in the image. There will be four fiducial-containing nanowells in the shape of a square, as
shown in Figure 23 (above). Position 143 is located at the lower right corner of the stage fixture.
10. Select DAPI as the illumination channel for a second time.
11. Examine the field of view for Pos143 to confirm that you are satisfied with the focus. If not, check
that the chip is sitting perfectly flat on the microscope’s Chip Holder.
12. Close the Stage Position List window.
13. Under the Channels table of the Multi-Dimensional Acquisition window, ensure that the following
stains are selected (preconfigured): DAPI (exposure = 200), Texas Red (exposure = 300). Texas Red
has excitation and emission wavelengths similar to propidium iodide.
Figure 24. Checking cell-stain channels.
Filter colors are assigned to each channel for easy identification of the cells:
•DAPI (teal) indicates the presence of the cell.
•Texas Red (red) indicates cells with membrane leakage.
14. Enter a short experiment name after the file name prefix (..\WafergenData\<experiment
name>) and the Chip Number (if you did not scan the chip earlier).
Figure 25. Entering chip number and directory location for images.

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C. Acquiring Images
1. Click Aquire! and accept the prompt to create a new folder based on the current date. The camera
will take images of the fields of view with both the DAPI and Texas Red filters. The entire image
folder should contain 288 TIFF files from 144 fields of view (Pos0–Pos143); each field of view has
two sets of exposures, DAPI and Texas Red.
Figure 26. Acquisition of field of view images with DAPI and Texas Red filters.
2. After imaging is complete, the software will provide a prompt for converting the file. Click Yes. This
will launch the CellSelect software. See the CellSelect Software User Manual for more information.
D. System Shutdown and Next Steps
1. Turn off the Mercury Burner Power Supply, the Filter Controller, the Stage Controller, and the
Camera (refer to Figures 4–6 in Section III.A, above, for the location of each unit’s on/off switch).
2. Reapply the peeled release liner onto the top side of the double-sided intermediate film on the chip.
3. Place the imaged chip into an empty Chip Holder that has been prechilled at –80°C (Figure 27,
below). Make sure that the imaging film is well sealed over the chip. The Chip Holder should click
closed, indicating a proper magnetic seal.
Figure 27. Chip Holder.
4. Freeze the cells at –80°C for a minimum of 10 minutes before proceeding to the next step. Refer to
the protocol or user manual specific to your application for more information.

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Appendix A: Maintenance and Troubleshooting
Maintenance
CAUTION: Use of equipment and reagents for cell preparation and isolation with
the ICELL8 Single-Cell System may cause exposure to toxic or biohazardous
chemicals, thereby presenting a hazard. Always wear appropriate personal protective
equipment (PPE), which should at minimum include gloves, eye protection, and a
lab coat, before cleaning the ICELL8 Imaging System.
1. After removing the chip from the stage, inspect the stage for any debris.
2. Wipe the stage and sides of the microscope ocular tube with 70% isopropanol alcohol. Avoid
touching the lens in the ocular tube.
3. Disinfect the bench or tabletop in accordance with your laboratory’s cleaning procedures. Make sure
that the overspray does not contact the lens in the microscope tube.
NOTE: If the lens in the ocular tube requires cleaning, use recommended laboratory procedures for
cleaning microscope lenses.
4. Refer to individual component user manuals for detailed information about maintenance and service
procedures.
Troubleshooting
NOTE: Refer to individual component user manuals for detailed information about maintenance and
service procedures.
Problem
Product
Software generates a
hardware error at startup.
Shut down computer, camera, and controllers. Restart
following the standard procedure. Ensure that the
component is turned on and warmed up. Check cable
connections to each hardware component.
Live window does not show
an image.
Check mechanical shutter, it should be in the Open
position (O).
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