Molecular Devices ImageXpress Pico Manual

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October 2019
ImageXpress®Pico
Automated Cell Imaging System
with CellReporterXpress® Software Version 2.5
Calibration Kit Guide

ImageXpress Pico Automated Cell Imaging System Calibration Kit Guide
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©2019Molecular Devices, LLC.
All rights reserved.

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Contents
Chapter 1: ImageXpressPicoAutomated Cell Imaging System 5
Obtaining Support 6
Product Documentation 7
About This Guide 7
Chapter 2: Package Contents 9
Chapter 3: Objective Maintenance 11
Installing an Objective 12
Calibrating an Objective 16
Adjusting an Objective Correction Collar 18
Cleaning an Objective 23
Chapter 4: Filter Cube Maintenance 25
Installing a Filter Cube 25
Calibrating a Filter Cube 29
Appendix A: Optional Parts and Accessories 31
Objectives 31
Filter Cubes 31
Accessories 32

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Chapter 1: ImageXpressPicoAutomated Cell Imaging
System
The Molecular Devices®ImageXpress® PicoAutomated Cell Imaging System is an all-in-one
platform for automatically acquiring and analyzing images from fluorescently labeled
biological samples in plates and slides. It enables you to increase the throughput of your
image acquisition and analysis, allowing you to gain insights in minutes. With the addition of
a modular option, the system provides environmental control for live cell imaging.
The core hardware component of the imaging system is a custom-designed, fully automated,
epi-illumination fluorescence microscope. The rapid autofocus and precision sample
movement features of the microscope allow large numbers of high-resolution images to be
acquired in the shortest possible time. All key optical and mechanical elements are
motorized, which allows complete control of the instrument configuration.
When used in combination with the CellReporterXpress® Image Acquisition and Analysis
Software, which features powerful image analysis capabilities and more than 25 available
predefined experimental protocols, the ImageXpressPico system becomes an extremely
flexible device, ideally suited for user-defined, automated assays.
Key components of the instrument include the following:
Built-in, internal light source comprised of six high-powered LEDs enables very high
sensitivity fluorescent imaging. Additional LED-based light sources allow transmitted light
and overview imaging.
High-sensitivity, 5-megapixel CMOS camera.
Hardware-based autofocus system with precision motorized Z-stage focus.
High-transmission fluorescence imaging optics with world-class chromatic aberration
correction, resolution, and image flatness.
Secondary camera provides overview imaging for slides.
Precision motorized sample (X-Y) stage.
High-quality Leica objectives in a six-position turret.
Filter cubes in a six-position turret.
Motorized selection of stage position, filter cubes, and objectives.
Temperature control up to 40°C (104° F) for live cell imaging.
Optional environmental control system, which enables you to regulate humidity, CO2,
and O2inside the environmental control cassette for multi-day, live-cell, time-lapse
imaging.
Operation and configuration control by the integrated CellReporterXpress software.
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Obtaining Support
Molecular Devices is a leading worldwide manufacturer and distributor of analytical
instrumentation, software, and reagents. We are committed to the quality of our products
and to fully supporting our customers with the highest level of technical service.
Our Support website, www.moleculardevices.com/service-support, describes the support
options offered by Molecular Devices, including service plans and professional services. It
also has a link to our Knowledge Base, which contains documentation, technical notes,
software upgrades, safety data sheets, and other resources. If you still need assistance after
consulting the Knowledge Base, you can submit a request to Molecular Devices Technical
Support.
Technical Support
You can contact Molecular Devices Technical Support by submitting a support request
through the Knowledge Base or by phone. To find regional support contact information, visit
www.moleculardevices.com/contact.
You need the instrument serial number.
The serial number is located on the back panel of the instrument.

Chapter 1: ImageXpressPicoAutomated Cell Imaging System
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Documentation
Review the product documentation on the Knowledge Base, including installation guides and
user guides. In addition, online Help is available within the CellReporterXpress software.
Press F1 to access Help for the current page.
Additional Resources
Web-based microscopy courses:
www.leica-microsystems.com/science-lab
www.ibiology.org/ibioeducation/taking-courses/ibiology-microscopy-short-course.html
The Molecular Probes Handbook offers advice on fluorescent probes and can help you
determine if there are better stains available for your analysis:
www.lifetechnologies.com/us/en/home/references/molecular-probes-the-
handbook.html
Product Documentation
The following guides are available on the Molecular Devices Knowledge Base at
mdc.custhelp.com:
CellReporterXpress Installation Guide
CellReporterXpress Release Notes
CellReporterXpress User Guide
ImageXpressPico Pre-Installation Guide
ImageXpressPico Installation Guide
ImageXpressPico User Guide
ImageXpressPico Calibration Kit Guide
In addition, the CellReporterXpress software includes context-sensitive Help that you can
access from within the software. Just press the F1 key from within the software to view Help
for the current page.
Tip: Molecular Devices recommends that you review the documentation before
installing or using the ImageXpressPico system or the CellReporterXpress software.
About This Guide
This guide is intended for the scientist who receives a calibration kit along with the
aftermarket purchase of an objective or filter cube for the ImageXpressPico system. It
describes how to install and calibrate the objective or filter cube.
The information in this guide is subject to change without notice. Molecular Devices
recommends that you review the guide on the Knowledge Base for the most up-to-date
information.

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Chapter 2: Package Contents
The Calibration Kit includes the following:
Item Part
number
Description
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9100-0099 Fluorescent Bead Slide
1-GP-7 Red Plastic Slide
1-GP-8 Green Plastic Slide
1-GP-11 Yellow Plastic Slide
For a complete list of the contents of the package, see the enclosed packing list.
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Chapter 3: Objective Maintenance
Objective maintenance procedures include the following:
Installing an Objective, see page 12
Calibrating an Objective, see page 16
Adjusting an Objective Correction Collar, see page 18
Cleaning an Objective, see page 23
You can identify the magnification of an objective by the color band:
Objective Magnification Color Band
4x Red
10x Yellow
20x Green
40x Light Blue
63x Dark Blue
The standard objectives in the ImageXpressPico system are configured and calibrated by
Molecular Devices when the instrument is delivered.
CAUTION! Replace objectives in their original positions.
Tip: Molecular Devices recommends removing and maintaining only one objective at
a time.
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Installing an Objective
Before installing an objective, review the following:
Access only the user-serviceable components inside the enclosure as described in the
procedure. Avoid contact with other components as they can be damaged or knocked
out of alignment.
To prevent dust from collecting inside the instrument, keep all access doors closed
unless you are performing maintenance tasks.
Ensure that all components and access doors are closed before starting the instrument.
CAUTION!
To prevent skin oils from damaging the optical coatings, Molecular Devices
recommends that you wear powder-free disposable gloves when handling
objectives and filter cubes.
With the instrument power on, do not manually rotate the objective turret.
Manually rotating the objective turret can damage the instrument.
Molecular Devices precalibrates the objectives to specific slots in the turret. You must install
the objectives as follows:
Slot Objective Magnification
1 4x
2 10x
3 20x
4 empty
5 40x or 63x
6 empty
Note:
Depending on how your ImageXpressPico system is configured, you may not
have all the objectives.
The 40x objective and the 63x objective cannot be installed in the instrument
simultaneously.
The 63x objective cannot be installed when the environmental control cassette is
loaded.

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Installing an Objective in an Empty Slot
You must install objectives in specific slots in the turret. See Installing an Objective on page
12 for details.
To install an objective in an empty slot:
1. In the CellReporterXpress software, on the Home page, click Devices.
2. Click Show Device Options to expand the details for the instrument where you
want to install an objective.
3. Click the Objectives tab.
4. In the tile for the objective slot where you want to install, click Component
Exchange.
5. Click the Choose Objective drop-down list box, and select the objective you want to
install.
6. Click Open Maintenance Door.
7. Install the objective in the slot by gently turning it clockwise.
CAUTION! Retain the objective case for future storage needs. When not installed
in the instrument, an objective should always be stored in its case.
8. Do both of the following:
a. Manually close the maintenance door.
b. In the CellReporterXpress software, click Close Maintenance Door.
9. Click Apply.
After you install a new objective from an after-sales purchase, you must calibrate it. See
Calibrating an Objective on page 16 for details.

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Replacing an Objective (Different Magnification)
You must install objectives in specific slots in the turret. See Installing an Objective on page
12 for details. Because of this, the only reason to replace an objective with another objective
of a different magnification is when you are a swapping the 40x objective and the 63x
objective in slot 5.
To replace an objective with an objective of a different magnification:
1. In the CellReporterXpress software, on the Home page, click Devices.
2. Click Show Device Options to expand the details for the instrument where you
want to replace an objective.
3. Click the Objectives tab.
4. In the tile for objective slot 5, click Component Exchange.
5. Click the Choose Objective drop-down list box, and select the objective you want to
install.
6. Click Open Maintenance Door.
7. Remove the objective to be replaced from the instrument by gently turning it
counterclockwise.
CAUTION! When not installed in the instrument, an objective should always be
stored in its case.
8. Install the replacement objective in the slot by gently turning it clockwise.
CAUTION! Retain the objective case for future storage needs. When not installed
in the instrument, an objective should always be stored in its case.
9. Do both of the following:
a. Manually close the maintenance door.
b. In the CellReporterXpress software, click Close Maintenance Door.
10. Click Apply.
After you replace an objective, you may need to calibrate it. See Calibrating an Objective on
page 16 for details.

Chapter 3: Objective Maintenance
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Replacing an Objective (Same Magnification)
You must install objectives in specific slots in the turret. See Installing an Objective on page
12 for details. The typical reason to replace an objective with another objective of the same
magnification is when you are replacing a damaged objective.
To replace an objective with an objective of the same magnification:
1. In the CellReporterXpress software, on the Home page, click Devices.
2. Click Show Device Options to expand the details for the instrument where you
want to replace an objective.
3. Click the Objectives tab.
4. In the tile for the objective slot where you want to install, click Component
Exchange.
5. Click the Choose Objective drop-down list box, and select None.
6. Click Open Maintenance Door.
7. Remove the objective to be replaced from the instrument by gently turning it
counterclockwise.
CAUTION! When not installed in the instrument, an objective should always be
stored in its case.
8. Install the replacement objective in the slot by gently turning it clockwise.
CAUTION! Retain the objective case for future storage needs. When not installed
in the instrument, an objective should always be stored in its case.
9. Do both of the following:
a. Manually close the maintenance door.
b. In the CellReporterXpress software, click Close Maintenance Door.
10. Click Close.
After you replace an objective with another objective, you may need to calibrate it. See
Calibrating an Objective on page 16 for details.
Note: If you re-install the same objective (after cleaning it, for example), you typically
do not need to calibrate it.

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Calibrating an Objective
After you install a new objective from an after-sales purchase, you must calibrate it. A
calibration kit, which is included with any after-sales objective purchase, includes the
following:
Fluorescent Bead Slide (9100-0099)
Red Plastic Slide (1-GP-7)
Green Plastic Slide (1-GP-8)
Orange Plastic Slide (1-GP-11)
You will need to provide the following item for the calibration process:
4-Slide Holder (5068503)
To calibrate an objective:
1. In the CellReporterXpress software, on the Home page, click Devices.
2. Click Show Device Options to expand the details for the device where you want to
calibrate an objective.
3. Click the Objectives tab.

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4. Note the status of the objective, which is indicated in the tile.
If the tile indicates Not Calibrated, continue to the next step to perform the
calibration procedure.
If the tile indicates Calibrated, you typically do not need to perform the calibration
procedure and you can skip this procedure.
Note: An exception to this is when you replace an objective with another
objective of the same magnification. In this case, you should continue to the next
step and perform the calibration procedure regardless of the label in the tile.
5. In the tile for the objective you want to calibrate, click Objective Calibration.
6. Follow the on-screen instructions to complete the calibration.
Tip:
In the Choose Slide Holder drop-down list box, select 4 Slide Holder.
Insert the slides face down in the slide holder (that is, printed side down).
When each step completes, click Next to continue to the next step.

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Adjusting an Objective Correction Collar
The 40x objective and 63x objective have application-optimized correction collars to
compensate for well bottom thickness or coverslip thickness. The collars have a range of
0mm to 2mm correction. Changing this setting adjusts the distances between components
inside the objective barrel. Image quality and resolution are very dependent on properly
setting these collars.
The settings to be used depend on the well bottom thickness of the plate or the coverslip
thickness on the slide on which the specimen is mounted. In general, set the correction collar
for the physical thickness of the plate or slide that you are imaging. The physical thickness
can be determined by the plate specifications from the plate manufacturer.
Note: Do not use a plate, slide, or coverslip with a thickness that is out of the range of
the correction collar for the selected objective.
Before adjusting an objective correction collar, review the following:
Access only the user-serviceable components inside the enclosure as described in the
procedure. Avoid contact with other components as they can be damaged or knocked
out of alignment.
To prevent dust from collecting inside the instrument, keep all access doors closed
unless you are performing maintenance tasks.
Ensure that all components and access doors are closed before starting the instrument.
CAUTION!
To prevent skin oils from damaging the optical coatings, Molecular Devices
recommends that you wear powder-free disposable gloves when handling
objectives and filter cubes.
With the instrument power on, do not manually rotate the objective turret.
Manually rotating the objective turret can damage the instrument.
You would typically adjust a correction collar as part of setting up an acquisition.

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Adjusting an Objective Correction Collar for a Plate
To adjust an objective correction collar for a slide:
1. In the CellReporterXpress software, on the Home page, click Acquisition.
2. Click Add Protocol.
3. Click New Slide Acquisition.
4. In the Available Acquisition Devices list, select the instrument.
5. On the left side of the screen under Steps, click Acquisition Settings.
6. On the right side of the screen under Tools, click Slide Format.
7. In the Slide Format list, select the slide format.
8. On the right side of the screen under Tools, click Objectives.
9. In the Objectives list, select the objective.
If a correction collar adjustment is required, the software displays the recommended
setting.
Note: When using the environmental control cassette with a 40x objective, you
must adjust the objective correction collar an additional 0.7 mm above the
recommended setting to account for the thickness of the cassette glass bottom.

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10. On the left side of the screen under Steps, click Acquisition Device.
11. On the right side of the screen, click Set Up for Adjustment of Objective Collar.
12. Click OK.
The objective door opens.
13. If needed, loosen the objective from the instrument by gently turning it
counterclockwise.
14. Rotate the correction collar to its new setting.
Tip: You might need a flashlight to see the markings for the graduated scale on
the barrel and its current setting.
15. If you loosened the objective, tighten it by gently turning it clockwise.
Note: When tightening the objective, take care to avoid changing the correction
collar setting.
16. Close the objective door.
17. Click OK.