Molecular Devices ImageXpress Confocal HT.ai User manual

5084773 A
March 2021
ImageXpress Confocal HT.ai
High-Content Imaging System
User Guide

ImageXpress Confocal HT.ai High-Content Imaging System User Guide
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Contents
Safety Information 7
Safety Symbols in This Guide 7
Safety Labels on the Instrument 8
Protective Housing and Safety Interlocks 9
Laser Safety 10
Electrical Safety 12
Moving Parts Safety 13
Lifting Hazard 13
Chemical and Biological Safety 14
Cleaning and Maintenance 14
Water Immersion Option Safety 15
Chapter 1: ImageXpress Confocal HT.ai System 17
ImageXpress Confocal HT.ai System Features 18
MetaXpress Software Features 21
Theory of Operation 22
Chapter 2: Using the ImageXpress Confocal HT.ai System 27
Starting the System 27
Understanding Status Lights 33
Maintaining the Instrument 34
Shutting Down the System 34
Chapter 3: System Installation and Testing 35
Verifying Device Settings in the Meta Imaging Series Administrator Software 36
Verifying Camera Settings in the Meta Imaging Series Administrator Software 41
Verifying and Backing Up Settings in the MetaXpress Software 42
Chapter 4: Preparing for Acquisition 53
Assay Design 53
Plate Selection 55
Sample Preparation 57
Plate Acquisition Settings 58
Instrument Maintenance 61
Chapter 5: Configuring Plate Acquisition Options 63
Accessing the Plate Acquisition Setup Dialog 64
Plate Acquisition Setup Dialog Layout 65

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Plate Acquisition Setup Dialog: Configure Tab, Objective and Camera Tab 69
Plate Acquisition Setup Dialog: Configure Tab, Plate Tab 72
Plate Acquisition Setup Dialog: Configure Tab, Sites to Visit Tab 77
Plate Acquisition Setup Dialog: Configure Tab, Acquisition Tab 90
Specifying the Number of Acquisition Wavelengths 95
Configuring Acquisition Wavelengths 96
Configuring Series Acquisition Options 106
Configuring Journals to Run During Acquisition 115
Configuring Post-Acquisition Analysis Options 118
Plate Acquisition Setup Dialog: Configure Tab, Display Tab 121
Viewing the Summary Panel 123
Saving Plate Acquisition Protocols 124
Running Plate Acquisition Protocols 125
Chapter 6: Customizing the MetaXpress Software 127
Users and Groups in the Meta Imaging Series Administrator Software 128
Custom Toolbars and Taskbars 131
Default Paths for Data 134
Chapter 7: Expansion Solution Options 135
Environmental Control Option 135
Transmitted Light Option 162
Water Immersion Option 179
Chapter 8: Maintenance 189
Maintenance Precautions 189
Routine Maintenance 190
Cleaning the Instrument 190
Light Source Maintenance 192
Replacing the Confocal Disk Box 192
Creating Hardware Profiles 198
Filter Maintenance 200
Objective Maintenance 209
Updating Legacy Shading Correction Settings 233
Turning Off the Status Indicator Lights 236
Obtaining Support 237

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Appendix A: Instrument Specifications 241
Appendix B: Compatible Objectives 243
Appendix C: Filter Specifications and Calibration Compatibility 247
Appendix D: Optional Parts and Accessories 249
Appendix E: File Privileges for the MetaXpress Software 251
Appendix F: Robotic Plate Handling 253
Verifying External Control Settings 254
Appendix G: Electromagnetic Compatibility 255

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Safety Information
This section describes the safe use of the instrument. Safety includes an understanding of the
information in this guide, the safety labels on the instrument, and the precautions that you must
follow before and during operation of the instrument.
It is important to confirm that everyone involved with the operation of the instrument has:
Received instruction in general safety practices for laboratories.
Received instruction in specific safety practices for the instrument.
Read and understood all Safety Data Sheets (SDS) for all materials being used.
Read and understood all instrument documentation, including all warnings and cautions.
Always remember that the key to safety is operating the instrument with care.
DANGER! If the instrument is used in a manner not specified by Molecular Devices, the
protection provided by the equipment might be impaired.
Safety Symbols in This Guide
All safety symbols in the user guide are framed within a triangle. An exclamation mark is used
for most safety symbols. Other symbols can warn of specific hazards, such as biohazard,
electrical, or laser safety warnings.
When danger, warning, or caution statements appear in this guide, be careful to follow the
specific safety information related to them.
The following safety symbols appear in this guide. Each symbol implies a particular amount of
observation or recommended procedure as described:
DANGER! A danger statement indicates a situation or operation that could cause
serious personal injury or death if precautions are not followed. The danger symbol
can vary depending on the hazard. The definition of the symbol is included in the text
of the statement.
WARNING! A warning statement indicates a situation or operation that could cause
personal injury if precautions are not followed. The warning symbol can vary
depending on the hazard. The definition of the symbol is included in the text of the
statement.
CAUTION! A caution statement indicates a situation or operation that could cause
damage to the instrument or loss of data if correct procedures are not followed.
In addition, the following user-attention symbols appear in this guide:
Note: A note calls attention to significant information.
Tip: A tip provides useful information or a shortcut, but is not essential to the completion
of a procedure.

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Safety Labels on the Instrument
Each safety label found on the instrument contains an alert symbol that indicates the type of
potential safety hazard related to the label. The following table lists the alert symbols that can
be found on Molecular Devices instruments.
Symbol Indication
Indicates that the product documentation must be consulted.
Indicates a potential laser hazard. The instrument is rated a
Class 4 high-power laser product. See Laser Safety on page
10.
Indicates a potential lifting hazard. See Instrument
Specifications on page 241 for details on the weight of the
instrument.
Indicates a potential pinch hazard.
Indicates a potential heat hazard.
Indicates power on. See Powering On the Instrument on page
27.
Indicates power off. See Shutting Down the System on page
34.
Table S-1: Safety Symbols

Safety Information
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Protective Housing and Safety Interlocks
The instrument features a protective outer housing and interlocks, which are designed to
protect you from exposure to laser light, hot surfaces, and moving parts.
The top door of the instrument is interlocked. Do not operate the instrument with the top door
open. Do not disable an interlock. When the top door is open, the laser light source is disabled
to prevent hazards associated with laser emission.
DANGER! Do not override any interlocks, open the protective housing, or attempt to
gain access to the interior of the instrument. Doing this can damage the instrument
components and result in hazardous exposure to laser light, hot surfaces, moving parts,
or high voltage.
Safety Interlock Failure
If the focusing laser stays on when the top door of the instrument is open, it is unsafe to
continue using the instrument due to a safety interlock failure. Contact Molecular Devices
Support immediately. See Obtaining Support on page 237.
Non-Interlocked Panels
The instrument has several panels that are intended for use by field service personnel only and
are not interlocked. All service panels are secured to the protective housing with screws and
require a special tool to remove.
WARNING! If you are instructed to remove non-interlocked panels, make sure that the
instrument is powered OFF and the power cable is unplugged. Never operate this
instrument with any covers or panels removed. Do not attempt to access the service-
only areas inside the instrument when the power cable is connected.

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Laser Safety
WARNING! LASER LIGHT. This symbol indicates that a potential hazard to personal
safety exists from a laser source. When this symbol appears in this guide, follow the
specific safety information related to the symbol.
The ImageXpress Confocal HT.ai system contains high-power laser products:
The external light source is a Class 4 high-power laser product.
The autofocus system uses a Class 3b high-power laser product.
Never attempt to open the instrument or access the high-power laser products. Installation and
service is to be performed by a Molecular Devices Field Service Engineer only.
The Class 4 laser product is designed and tested in accordance with IEC 61010-1, CAN/CSA
C22.2 Number 61010-1, CENELEC EN 61326-1:2006, and FCC Part 15 Subpart B, Class A.
Note: See www.89north.com/fluorescence-microscopy-products-by-brand/89-
north/ldi-laser-diode-illuminator for complete details on the external light source.
Item Description
Emitted Wavelength 405 nm - 730 nm
MaximumOutputPower 2 W
Laser Class Class4
Table S-2: External Laser Module Specifications (Light Source)
Item Description
Wavelength 690nm
Maximum OutputPower 20mW, continuous wave
Laser Class Class3b
Table S-3: Embedded Laser Module Specifications (Autofocus System)

Safety Information
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The ImageXpress Confocal HT.ai system is equipped with a redundant laser safety system.
When samples are being loaded or unloaded, hardware interlocks prevent the laser modules
from powering on until the instrument top door is closed.
DANGER! VISIBLE AND/OR INVISIBLE LASER RADIATION. Do not operate the
external light source when housing is open and interlocks are defeated. Avoid eye or
skin exposure to direct or scattered radiation.
WARNING! LASER LIGHT.
Do not attempt to repair or adjust the Class 4 high-power laser product. Removing
the top panel, safety interlocks, external light source, and microscope objective
and then looking into the Class 4 or Class 3b laser beam can cause severe eye
injury and blindness.
Operate the instrument only when all the doors and panels of the instrument are in
place and closed.
WARNING!
A Class 4 laser beam can cause materials to smolder or burn, especially at close
range. Dark materials, which absorb heat, and materials such as plastic, paper, and
fabric can easily be burned by visible laser beams.
Fumes produced when laser radiation vaporizes or burns a target material—
whether metallic, organic, or biologic—may be hazardous.
To avoid burning materials at close range, keep the laser beam moving and avoid
long exposures.
To avoid unintentional operation of the Class 4 laser beam, always power off the
external light source when not in use.

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Electrical Safety
To prevent electrically related injuries and property damage, inspect all electrical equipment
before use and immediately report all electrical deficiencies. Contact Molecular Devices
Technical Support to service equipment that requires the removal of covers or panels.
We recommend that you power off the instrument when it is not in use.
WARNING!
The ImageXpress Confocal HT.ai system is an Equipment Class 1 product that
relies on protective earth grounding for safe operation. Any interruption of the
protective earth ground conductor—inside or outside the instrument—or
disconnection of the protective earth ground terminal can result in personal injury.
Do not position the equipment so that it is difficult to operate the power switch on
the front of the ImageXpressSystems Power and Options Controller.
WARNING! HIGH VOLTAGE. Do not operate the external light source with the
external light source housing open. Never attempt to open or tamper with the laser
light source housing due to the extreme hazard of the Class 4 high-power laser
product.
ImageXpressSystems Power and Options Controller
A single power cable connects the instrument to the external ImageXpressSystems Power
and Options Controller. The external power controller has an input voltage rating of 100VACto
240VAC, 50/60Hz, 12amps maximum. The power controller contains no user-serviceable
parts.
WARNING! Before attempting to access any internal service areas of the instrument ,
unplug the power cable.
Fuses and Circuit Protection
In the ImageXpress Confocal HT.ai system, the ImageXpressSystems Power and Options
Controller provides over-current protection for the light source limited to 15amps maximum.
The power controller contains no user-serviceable parts.

Safety Information
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Moving Parts Safety
The instrument contains moving parts that can cause injury. Under normal conditions, the
instrument is designed to protect you from these moving parts.
To prevent injury:
Never try to exchange labware, reagents, or tools while the instrument is operating.
Never try to physically restrict the moving components of the instrument.
Avoid contact with the moving top door of the instrument is open.
Some user-controlled moving parts exist behind the access doors on the side of the
instrument. When doing maintenance operations that require access through these doors,
avoid contact with parts that are in motion.
WARNING! Do not attempt to access the interior of the instrument unless specifically
instructed to do so. The moving parts inside the instrument can cause injury. Do not
operate the instrument with any covers or panels removed.
Note: Observe all warnings and cautions listed for all external devices attached to or in
use during the operation of the instrument. See the applicable user guide for the
operating and safety procedures of that device.
Lifting Hazard
WARNING! LIFTING HAZARD. The ImageXpress Confocal HT.ai system weighs
approximately 104kg (230lb). Do not attempt to lift or move the instrument without
assistance.
CAUTION! Moving the instrument can disrupt sensitive optical alignments. We
recommend that you contact Molecular Devices Technical Support to schedule a Field
Service Engineer to help with moving your instrument. Your warranty or service contract
does not cover problems caused during or as a result of shipment or relocation of the
instrument.

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Chemical and Biological Safety
Normal operation of the instrument can involve the use of materials that are toxic, flammable,
or otherwise biologically harmful. When using such materials, observe the following
precautions:
Handle infectious samples based on good laboratory procedures and methods to prevent
the spread of disease.
Observe all cautionary information printed on the original containers of solutions before
their use.
Dispose of all waste solutions based on the waste disposal procedures of your facility.
Operate the instrument in accordance with the instructions outlined in this guide, and take
all the required precautions when using pathological, toxic, or radioactive materials.
Splashing of liquids can occur. When working with potentially hazardous liquids, take
applicable safety precautions, such as wearing safety glasses and protective clothing.
Observe the applicable cautionary procedures as defined by your safety officer when
using hazardous materials.
Observe the applicable cautionary procedures as defined by your safety officer when
using flammable solvents in or near an instrument that is powered on.
Observe the applicable cautionary procedures as defined by your safety officer when
using toxic, pathological, or radioactive materials.
WARNING! Never use the instrument in an environment where potentially damaging
liquids or gases are present.
Cleaning and Maintenance
Observe the cleaning procedures outlined in this guide for the instrument.
Do the following before you clean equipment that has been exposed to hazardous material:
Contact the applicable Chemical and Biological Safety personnel.
Review the Chemical and Biological Safety information contained in this guide. See
Chemical and Biological Safety on page 14.
Perform only the maintenance tasks described in this guide. Contact Molecular Devices to
inspect and perform preventive maintenance service on the instrument each year. See
Obtaining Support on page 237.
WARNING! BIOHAZARD. It is your responsibility to decontaminate components of
the instrument before you return parts to Molecular Devices for repair. Molecular
Devices does not accept items that have not been decontaminated where it is
applicable to do so. If parts are returned, they must be enclosed in a sealed plastic bag
stating that the contents are safe to handle and are not contaminated.
For approved cleaning and maintenance procedures, see Maintenance on page 189.

Safety Information
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Water Immersion Option Safety
If your ImageXpress system includes the Water Immersion option, review the following
information to ensure proper operation and to reduce the risk of damage to the instrument and
components.
CAUTION!
Do not remove or change the water immersion objectives that are installed within
the instrument. A Molecular Devices Field Service Engineer must install and remove
water immersion objectives.
Do not touch the leak detection sensor, which is the brass color ring around the top
of the objective below the cap.
Do not place the water immersion controller on top of the instrument or near
electronics. The water immersion controller is not watertight. Water will not be
contained within the controller if water spills into the water immersion controller.
Do not move the water immersion controller to a location that is above or below
where it is installed by the Field Service Engineer. To ensure high-performance
acquisition with water immersion, the Field Service Engineer calibrates the water
dispense rate. If you change the elevation of the water immersion controller, you
can negatively affect the calibration.
Do not attempt to remove the water immersion controller cover. With the exception
of the water bottles, the controller does not contain any user-serviceable parts.
Use only distilled water in the source water bottle. Deionized water can be used,
but is not required. Do not use tap water, and do not reuse the water that is in the
waste bottle.

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Chapter 1: ImageXpress Confocal HT.ai System
The ImageXpress® Confocal HT.ai High-Content Imaging System is an integrated cellular
imaging and analysis system designed for rapid, automated screening of fluorescently labeled
biological samples in microplates. With the addition of modular options, the system provides
environmental control for live cell imaging and uses transmitted light capability for label-free
imaging.
The core hardware component of the imaging system is a custom-designed, fully automated,
epi-illumination fluorescence, confocal 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,
with asynchronous command execution, allowing complete real-time control of the instrument
configuration through the MetaXpress® High-Content Image Acquisition and Analysis Software.
When used in combination with the powerful image analysis capabilities of the MetaXpress
software, the instrument becomes an extremely flexible and programmable device, ideally
suited for user-defined, high-speed automated assays.
Key components of the instrument include the following:
External solid-state laser light source with seven channels connected by a fiber-optic light
guide and selectable dichroic and emission filters inside the instrument
ScientificCMOS camera
Laser autofocus system with precision motorized Z (focus) stage
Image-based autofocus
Precision motorized X-Y (sample) stage
User-changeable, high-quality, Nikon objectives in a four-position linear selector
User-changeable and selectable emission and dichroic filters in two separate controllable
wheels inside the confocal module
Spinning confocal single-disk box with available dual-disk boxes designed for confocal
screening and confocal research
Selectable binning modes to decrease exposure time and increase throughput
Motorized selection of stage position, filters, and objectives with asynchronous operation
High-transmission fluorescence imaging optics with world-class chromatic aberration
correction, resolution, and image flatness
Operation and configuration control by the integrated MetaXpress software
Optional expansion solutions available for environmental control, phase contrast
transmitted light, and water immersion objectives
Optional plate-handling robot with an automated barcode reader
1

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ImageXpress Confocal HT.ai System Features
Figure 1-1: ImageXpress Confocal HT.ai SystemComponents without Options
Illumination System: Excitation
The ImageXpress Confocal HT.ai system is equipped with an external light source connected
to the instrument with a light guide.
External Light Source
The external light source is a Class 4 high-power laser product with a rated lifetime of 20,000
hours. There are no user-replaceable parts in the solid-state light source.
DANGER! See Laser Safety on page 10 for details on the Class 4 high-power laser
product and specific warning and danger statements for working with the external
laser light source.
Light Source Filter Wavelength Ranges
Filter Wavelength
DAPI 405/20nm
CFP 445/20 nm
FITC 467.5/21 nm
YFP 520/10 nm
TRITC and Texas Red 555 nm
Cy5 638/17 nm
Cy7 725/30 nm
Table 1-1: ImageXpress Confocal HT.ai System Light Source Excitation Spectra
Illumination Optics
The output end of the fiber optic light guide is imaged onto the sample by a set of internal
optics and the objective to provide bright and uniform illumination of the specimen over a
wide field of view.

Chapter 1: ImageXpress Confocal HT.ai System
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Filter Changer
The 8-position emission filter wheel uses standard, commercially available filters. Molecular
Devices offers and recommends Semrock filters, based on ImageXpress Confocal HT.ai
system validation testing.
The 5-position dichroic filter wheel uses a unique frame. You must order the dichroic mirror
filters through Molecular Devices.
Objective (Z) Stage
Motorized Z Stage
A linear encoder that features better than 100nm resolution monitors the Z stage position.
Objectives
The standard objectives are Nikon CFI60 series. The objective lens focuses excitation light
onto the sample and collects light that the sample emits. See Compatible Objectives on page
243.
CAUTION! You must contact Molecular Devices Technical Support to schedule a Field
Service Engineer to install or remove a water immersion objective.
Motorized Objective Changer
The instrument includes a 4-position objective changer. Only the selected objective moves up
and down when you change the position.
Sample (X-Y) Stage
Sample
The plate holder is designed to scan multi-well microplates in standard ANSI (SBS) formats with
plastic or glass bottoms. It can accommodate other plate formats that have standard
microplate footprint dimensions. For example, you can use the glass slide adapter (included in
the accessory kit) to image glass slides. Optimal image quality depends on plate flatness, well
bottom thickness, and optical clarity.
Plate Holder and Plate Clamp
A spring-loaded mechanical clamp holds the sample plate securely in the plate holder. The
clamp automatically opens when the X-Y stage moves to the load/eject position, and
automatically closes when the X-Y stage moves the plate into position for imaging.
Motorized X-Y Stage
A linear encoder that features better than 100nm resolution monitors the X-Y stage position.
Autofocus Laser
The ImageXpress Confocal HT.ai system uses a laser to autofocus the image. The red
(690nm) diode laser projects a laser spot onto the sample. Reflections of this spot from the
bottom of the plate and the plate-sample interface are imaged by a dedicated, fast-focus
sensor and the spot acts as a reference for focus software.

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Imaging System: Emission
Tube Lens
The tube lens collects collimated light from the objective and focuses it onto the detector
plane of the camera. The emission wavelength range is 400nm to 750nm.
Camera
The ImageXpress Confocal HT.ai system uses a scientific CMOS camera with a 2048×2048
image sensor format (6.5×6.5µm pixel size) and a peak quantum efficiency of 82%.
Confocal Disks
Three Confocal disk box configurations are available for the ImageXpress Confocal HT.ai
system. For the confocal performance details with different objectives, see Compatible
Objectives on page 243.
Disk Part Number Use
5039730 Confocal Single-Disk Unit
(standard)
For basic imaging
5039731 Confocal Screening Dual-Disk Unit
(optional)
For basic and high-throughput imaging
5040287 Confocal Research Dual-Disk Unit
(optional)
For basic and high-resolution imaging
Table 1-2: Confocal Disks Geometries
Electronics
The base ImageXpress Confocal HT.ai system includes the following components:
ImageXpress Confocal HT.ai instrument
ImageXpressSystems Power and Options Controller, with cables
Acquisition computer, with monitor and cables
Solid-state laser light source, with fiber-optic light guide and cables
USB 2.0 cable for acquisition computer to instrument (camera control) and instrument to
acquisition computer (data)
USB 3.0 cable for internal camera to acquisition computer