ImageXpress Velos Laser Scanning Cytometer User Guide

Transcription

ImageXpress® Velos
Laser Scanning Cytometer
User Guide
0270-00007 D
May 2011
This document is provided to customers who have purchased Molecular Devices, Inc.
(“Molecular Devices”) equipment, software, reagents, and consumables to use in the
operation of such Molecular Devices equipment, software, reagents, and
consumables. This document is copyright protected and any reproduction of this
document, in whole or any part, is strictly prohibited, except as Molecular Devices
may authorize in writing.
Software that may be described in this document is furnished under a license
agreement. It is against the law to copy, modify, or distribute the software on any
medium, except as specifically allowed in the license agreement. Furthermore, the
license agreement may prohibit the software from being disassembled, reverse
engineered, or decompiled for any purpose.
Portions of this document may make reference to other manufacturers and/or their
products, which may contain parts whose names are registered as trademarks and/or
function as trademarks of their respective owners. Any such usage is intended only
to designate those manufacturers' products as supplied by Molecular Devices for
incorporation into its equipment and does not imply any right and/or license to use
or permit others to use such manufacturers' and/or their product names as
trademarks.
Molecular Devices makes no warranties or representations as to the fitness of this
equipment for any particular purpose and assumes no responsibility or contingent
liability, including indirect or consequential damages, for any use to which the
purchaser may put the equipment described herein, or for any adverse circumstances
arising therefrom.
For research use only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of Molecular Devices, Inc. or their
respective owners. These trademarks may not be used in any type of promotion or
advertising without the prior written permission of Molecular Devices, Inc.
TWISTER is a registered trademark of Caliper Life Sciences, Inc.
Product manufactured by Molecular Devices, Inc.
1311 Orleans Drive, Sunnyvale, California, United States of America 94089.
Molecular Devices, Inc. is ISO 9001 registered.
© 2011 Molecular Devices, Inc.
All rights reserved.
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purpose of This User Guide . . . . . . . . . . .
Conventions Used in This User Guide . . . .
Safety Information . . . . . . . . . . . . . . . . .
General Safety . . . . . . . . . . . . . . . . . . .
Lifting Hazard . . . . . . . . . . . . . . . . . . .
High-Voltage Hazard. . . . . . . . . . . . . . .
Laser Safety . . . . . . . . . . . . . . . . . . . .
Electrical Safety . . . . . . . . . . . . . . . . . .
Mechanical Safety . . . . . . . . . . . . . . . .
Hazardous Material Precautions . . . . . . .
Safety Labels. . . . . . . . . . . . . . . . . . . . .
Warning Labels . . . . . . . . . . . . . . . . . .
Maintenance and Service . . . . . . . . . . . .
Obtaining Support . . . . . . . . . . . . . . . . .
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Chapter 1 Introduction . . . . . . . . . . . . . . . . .
ImageXpress Velos Laser Scanning Cytometer
Overview . . . . . . . . . . . . . . . . . . . . . . . . . .
How the ImageXpress Velos
Laser Scanning Cytometer Operates. . . . . . . .
Instrument Specifications . . . . . . . . . . . . . . .
Connecting the ImageXpress Velos Cytometer
to the Computer . . . . . . . . . . . . . . . . . . . . .
Starting the Instrument and Software . . . . . .
0270-00007 D
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Contents
Chapter 2 Creating a Method . . . . . . . . . . . . . . . . .
Verify Emission Filters . . . . . . . . . . . . . . . . . . . . . .
For Anisotropy Assays . . . . . . . . . . . . . . . . . . . . .
Loading a Microplate into the ImageXpress Velos
Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the Plate Map . . . . . . . . . . . . . . . . . . . . .
Loading the Microplate . . . . . . . . . . . . . . . . . . . .
Steps to Create a Method . . . . . . . . . . . . . . . . . . .
Step 1: Set the scan parameters . . . . . . . . . . . . .
Step 2: Set the detect parameters . . . . . . . . . . . .
Step 3: Set the analysis parameters . . . . . . . . . . .
Step 4: Optimizing Settings in Calibration Mode . . .
Step 5: Select the wells to scan . . . . . . . . . . . . . .
Step 6: Save the method. . . . . . . . . . . . . . . . . . .
Using an Existing Method . . . . . . . . . . . . . . . . . . .
Step 1: Open a method and load a plate with
samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 2: Open the calibration screen and view
the real-time signal. . . . . . . . . . . . . . . . . . . . . . .
Step 3: Select the detector channel(s) and
plate column or well to display in the scope window
Step 4: Adjust the scan line position to find the
best objects or region to focus on . . . . . . . . . . . . .
Step 5: Adjust the collection or scanning focus. . . .
Step 6: Adjust the signal amplitude . . . . . . . . . . .
Step 7: Save the optimized parameter settings
with the method . . . . . . . . . . . . . . . . . . . . . . . . .
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Chapter 3 Creating a Process . . . . . . . . . . . . . . . . . . . .
Steps to Create a Process . . . . . . . . . . . . . . . . . . . . . . .
Step 1: Open image data (.bbd) . . . . . . . . . . . . . . . . .
Step 2: Specify a region of interest (ROI)
(optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 3: Set the brightness and contrast . . . . . . . . . . . .
Step 4: Open the analysis processing control panel . . . .
Step 5: Optimizing the intensity threshold . . . . . . . . . .
Step 6: Choose any additional processing and
anisotropy factors . . . . . . . . . . . . . . . . . . . . . . . . . . .
Step 7: Set the particle filters . . . . . . . . . . . . . . . . . . .
Step 8: Test and optimize the process . . . . . . . . . . . . .
Step 9: Save the process . . . . . . . . . . . . . . . . . . . . . .
Viewing Processed Well Data . . . . . . . . . . . . . . . . . . . . .
Viewing the Particle Summary . . . . . . . . . . . . . . . . . . . .
Customizing the Well Results. . . . . . . . . . . . . . . . . . . . .
Saving Analysis Results . . . . . . . . . . . . . . . . . . . . . . . .
Manually Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . .
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Chapter 4 Acquire and Save Images . . . .
Scanning a Plate and Viewing Images . . .
Starting a Scan . . . . . . . . . . . . . . . . . .
Saving Image Data . . . . . . . . . . . . . . . .
To save image data:. . . . . . . . . . . . . . .
Discarding Data . . . . . . . . . . . . . . . . . . .
Exporting Data . . . . . . . . . . . . . . . . . . .
Exporting Multiple Graphic Files . . . . . . .
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Chapter 5 Viewing Data Files. . . . . . . . . . . . . . .
Operating in Auto-analysis Mode . . . . . . . . . . . .
Viewing Results . . . . . . . . . . . . . . . . . . . . . . . .
Changing the Result Displayed in the Plate Map .
Saving the Image Data . . . . . . . . . . . . . . . . . . .
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Contents
Chapter 6 ImageXpress Velos Cytometer
Data Acquisition Quick Start Guide. . . . . . . . . . . .
Step 1: Verify Filters . . . . . . . . . . . . . . . . . . . . . .
Step 2: Calibration (optimize PMT & focus settings)
Step 3: Acquire and process data simultaneously in
ImageXpress Velos . . . . . . . . . . . . . . . . . . . . . . .
Appendix A ImageXpress Velos Software
Menu Commands and Toolbar. . . . . . . . . . . .
Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . .
File Menu. . . . . . . . . . . . . . . . . . . . . . . . . .
Image Menu . . . . . . . . . . . . . . . . . . . . . . .
Tool Menu . . . . . . . . . . . . . . . . . . . . . . . . .
Analysis Menu . . . . . . . . . . . . . . . . . . . . . .
View Menu. . . . . . . . . . . . . . . . . . . . . . . . .
Scan Menu. . . . . . . . . . . . . . . . . . . . . . . . .
Data Menu . . . . . . . . . . . . . . . . . . . . . . . . .
Window Menu . . . . . . . . . . . . . . . . . . . . . .
Autorun Menu . . . . . . . . . . . . . . . . . . . . . .
Control Menu . . . . . . . . . . . . . . . . . . . . . . .
Help Menu . . . . . . . . . . . . . . . . . . . . . . . . .
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Appendix B ImageXpress Velos Cytometer Files . . . . . 111
Appendix C Validated Fluorophores and
ImageXpress Velos Filters . . . . . . . . . . . . . . . . . . . . . . 115
Appendix D Resolution and Typical Scan Times . . . . . . 119
Appendix E Working in Autorun Mode . . . . .
Setting Up an Autorun with the Twister II
Microplate Handler . . . . . . . . . . . . . . . . . . .
Setting Up an Autorun to Do Multiple Scans. .
Setting Up Autorun with a Simulated Robot . .
Starting an Autorun . . . . . . . . . . . . . . . . . .
Pausing an Autorun . . . . . . . . . . . . . . . . . .
Aborting an Autorun . . . . . . . . . . . . . . . . . .
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Appendix F ImageXpress Velos Image Registration
Alignment Procedure . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing for the New Alignment Plate . . . . . . . . . . . . .
Preparing for Calibration . . . . . . . . . . . . . . . . . . . . . . .
Creating a Method for Alignment Plate . . . . . . . . . . . .
Well Alignment Calibration of Laser 1 (488 nm) . . . . . . .
Image Adjustment with the Alignment Plate . . . . . . . . .
Well Alignment Calibration of Laser 2
(405, 440, 532, or 640 nm wavelength) . . . . . . . . . . . .
Appendix G ImageXpress Velos Image Registration
Alignment Procedure with Bead Plate
(Legacy Plastic Plate) . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing for Calibration . . . . . . . . . . . . . . . . . . . . . . .
Well Alignment Calibration of Laser 1 (488 nm) . . . . . . .
Image Adjustment with Bead Plate . . . . . . . . . . . . . . .
(405, 440, 532, or 640 nm wavelength) . . . . . . . . . . . .
Appendix H ImageXpress Velos Intensity
Calibration Procedure . . . . . . . . . . . . . . . . . . . .
Preparing for Calibration . . . . . . . . . . . . . . . . . .
Intensity Calibration (ILUT) for Laser 1
(typically 488 nm) . . . . . . . . . . . . . . . . . . . . . .
Testing the ILUT Correction in Channels 1 and 2
Testing ILUT from Calibration . . . . . . . . . . . . .
Creating a Method for the Standard Uniformity
Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intensity Calibration (ILUT) for Laser 2. . . . . . . .
Appendix I Creating a Plate Configuration File
Preparation . . . . . . . . . . . . . . . . . . . . . . . . . .
Creating or Modifying a Plate File . . . . . . . . . . .
Adjusting the Well Alignment. . . . . . . . . . . . .
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Appendix J Maintenance . . . . . . . . . . . . . . . . . . . . . . . 195
Cleaning the Optics . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Transfer Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
0270-00007 D
7
Contents
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
8
0270-00007 D
Preface
Topics in this section:
• Purpose of This User Guide on page 9
• Conventions Used in This User Guide on page 9
• Safety Information on page 10
• Safety Labels on page 16
• General Safety on page 11
• Maintenance and Service on page 17
• Obtaining Support on page 18
This foreword provides information about the ImageXpress Velos Laser
Scanning Cytometer User Guide and important safety information on
the instrument.
Purpose of This User Guide
This user guide provides details on the technical features and
specifications of the ImageXpress® Velos Laser Scanning Cytometer. It
explains how to acquire and analyze data, and includes calibration and
maintenance instructions.
Conventions Used in This User Guide
This guide uses the following conventions:
WARNING! Indicates a possibility of severe or fatal injury to
the user or other persons if the precautions are not observed.
CAUTION! Indicates that damage to the instrument, loss of data, or
individual injury could occur if the user fails to comply with the advice
given.
Note: Provides additional significant information about a procedure or
description.
0270-00007 D
9
Preface
Tip! Provides useful information or shortcuts, but the information is not
essential to complete a procedure.
Safety Information
Safe operation is the responsibility of the end user. Read and
understand this user guide to avoid hazards prior to operating the
ImageXpress Velos Laser Scanning Cytometer. If you have questions or
are uncertain about any information, contact Molecular Devices
Technical Support or an authorized service provider before operating or
obtaining service for the instrument.
Make sure that you follow the precautionary statements presented in
this guide. Before using or servicing the ImageXpress Velos Laser
Scanning Cytometer, you must be familiar with the operation and
potential hazards of the instrument. You should read, understand, and
obey all safety precautions. Failure to follow these safety precautions
could result in serious personal injury or damage to the instrument.
Warnings in this document and labels on the instrument use
international symbols.
The operator of the ImageXpress Velos Laser Scanning Cytometer must
be trained in the correct operation of the instrument and in the safety
procedures. Using controls, making adjustments, or performing
procedures other than those specified in this guide can result in
hazardous exposure to laser light, high voltage, hot surfaces, or
moving parts. Exposure to these hazards can cause severe or fatal
injury.
Note: These safety practices are intended to supplement your national
and local health and safety regulations and laws. The information
provided covers instrument-related safety regarding the operation of
the instrument. The information does not cover every safety procedure
that should be practised. Ultimately, you and your organization are
responsible for compliance with national and local EHS (Environmental
Health and Safety) legal requirements and for maintaining a safe
laboratory environment.
10
0270-00007 D
General Safety
•
Place the laser scanner on a stable, level surface capable of
supporting a minimum of 180 pounds (81.8 Kg). Do not place
the instrument on an unstable surface. If the instrument is not
properly supported, serious damage or injury can occur.
• Locate the instrument in an area with adequate airspace for
ventilation.
• Never set a container of liquid on top of the instrument.
If any of the following occur, unplug the instrument and contact
Molecular Devices:
• The instrument has been dropped or damaged.
• Liquid has spilled inside the instrument.
• The power cord has been damaged or is frayed.
WARNING! Use the ImageXpress Velos Laser Scanning
Cytometer only as instructed in this guide. Do not attempt to
service the instrument. Only qualified service personnel
approved by Molecular Devices are authorized to service the
instrument. Do not remove the instrument case. If the
instrument case is removed, laser and electrical shock hazards
are exposed.
Lifting Hazard
WARNING! Two people are required to lift the instrument. Do
not attempt to lift or move the instrument without assistance.
The ImageXpress Velos instrument weighs approximately 130
pounds (59 kg).
CAUTION! Moving your instrument can disrupt sensitive optical
alignments. Contact technical support to schedule an FSE (field service
engineer) to help with moving your instrument. Your warranty or
service contract will not cover problems caused during or as a result of
shipment or relocation.
0270-00007 D
11
Preface
High-Voltage Hazard
Where present, the hazardous voltage label (see Table P-2), indicates a
potential electrical hazard. Do not attempt to open or service the
instrument’s power supply at any time.
Laser Safety
The ImageXpress Velos Cytometer can be equipped with a single laser
or a combination of a 488 nm laser and one of the following solid-state
lasers: 405, 440, 532, or 640 nm. The instrument design includes a
safety shutter and protective covers so that no laser light leaves the
instrument during operation. Never operate the instrument with the
laser exposed. An object in the direct path of the laser beam can be
damaged or injured. When used in accordance with the instructions in
this guide, the ImageXpress Velos Laser Scanning Cytometer is a Class
1 laser product per 21 CFR 1040.
WARNING! The ImageXpress Velos Laser Scanning Cytometer
is a Class 1 laser product when the laser is enclosed in the
instrument case. The laser is a Class 3B laser product with a
maximum output shown in Table P-1. If the instrument is not
operated according to the instructions in this guide, the laser
can cause serious damage or injury.
Table P-1 Embedded laser classification and power
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Nominal
Wavelength
(nm)
Typical
Power
(mW)
Maximum
Laser
Duration
Power (nm)
Embedded
Laser Class
405
50
90
Continuous
Wave
Class 3b
440
40
70
Continuous
Wave
Class 3b
488
20
70
Continuous
Wave
Class 3b
532
50
70
Continuous
Wave
Class 3b
640
40
500
Continuous
Wave
Class 3b
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WARNING! If the cover is removed and the interlock is
defeated when the instrument is turned on, laser radiation is
present. Only qualified, authorized personnel should install,
maintain, or operate this instrument.
The following warnings and cautions represent typical situations that
require special attention. Your knowledge and experience with your
specific environment must be also taken into consideration in order to
help ensure safety for personnel and equipment.
• The system must be installed and serviced by a Molecular
Devices Field Service Engineer (FSE).
• All instrument panels must be in place on the instrument while
the ImageXpress Velos Cytometer is operating.
• Do not remove safety labels or disable safety interlocks.
• Be aware of the potential hazards in the environment where the
equipment will be installed.
• Service personnel should wear laser safety eye protection while
working on the automation, laser, optical, electrical, and
electronics.
• Do not look directly into the laser beam.
WARNING! OCULAR HAZARD. Maintenance and servicing of the
lasers must only be done by personnel trained by Molecular
Devices. Visible Class 3b laser radiation is accessible with the
covers removed and the interlocks defeated. Approved safety
eye protection, rated for use with the emitted wavelength,
must be worn.
•
•
0270-00007 D
Replace all covers and safety shields after completing system
start up, troubleshooting, or maintenance procedures.
Pay careful attention to the safety labels on the instrument that
indicate where there is a potential for injury.
13
Preface
Figure P-1 Safety label: DANGER – Laser radiation
Where present, this label indicates there is a potential for exposure to
laser radiation.
Electrical Safety
The ImageXpress Velos Laser Scanning Cytometer can be configured
for any voltage from100 to 240 VAC nominal, 50 to 60 Hz. The system
is shipped for use with either 100–120 VAC (10A) or 200–240 VAC
(6A), but does not automatically switch between these voltages. Please
ensure that the unit is properly configured for the voltage applied to it;
otherwise it might not operate. The fuse ratings are 250VAC/6A or
120VAC/10A. If there are any questions concerning product
configuration, contact Molecular Devices.
CAUTION! The power supply cord is the primary way to disconnect the
instrument. Ensure that the instrument is connected to electrical power
with the power supply cord that is included with the instrument and
that the instrument is connected to a reliable ground source.
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Mechanical Safety
Figure P-2 Safety label: WARNING – Pinch point
Where present, this label indicates the presence of a potential
mechanical hazard during normal operation.
Hazardous Material Precautions
Use standard laboratory procedures and cautions when working with
chemicals.
WARNING! Always follow the manufacturer’s precautions
when working with chemicals. Molecular Devices is not
responsible or liable for any damages caused by, or as a
consequence of, the use of any hazardous material.
0270-00007 D
15
Preface
Safety Labels
If the safety label on the instrument becomes illegible or is missing for
any reason, contact Technical Support for a free replacement label.
While waiting for a replacement label, copy the label in Figure P-3 and
attach a copy of the label to the instrument.
.
Figure P-3 Safety label for instruments manufactured in the U.S. or
Singapore
16
0270-00007 D
Warning Labels
Table P-2 displays the safety symbols used on the ImageXpress Velos
Laser Scanning Cytometer and in this user guide.
Table P-2 Warning Labels
Safety
Symbol
Description
Safety
Symbol
Description
Electric Shock Hazard
Warning
Laser Hazard
Toxic Chemical Hazard
Lifting Hazard
Puncture Hazard
Maintenance and Service
User service and maintenance is strictly limited to the procedures
outlined in Maintenance on page 195. The majority of serviceable
components are accessed through the interlocked panels described
above and as such must be accessed by a Molecular Devices FSE. If
there is a problem or you have questions, contact Molecular Devices
Technical Support.
0270-00007 D
17
Preface
Obtaining Support
Part of effective communication with Molecular Devices is determining
the channels of support for the ImageXpress Velos system, including
the software. Molecular Devices provides a wide range of support
materials to help you troubleshoot any problems.
Complete the steps in order as detailed below.
1. Consult the documentation—Check the manual shipped with the
system.
2. Internet Support: Fill out the Technical Support Request Form at
http://www.moleculardevices.com/Support.html to send an
email to a pool of technical support representatives.
3. Call Customer Service: Contact Molecular Devices Customer
Service department at 800-635-5577 (United States only) or
+1-408-747-1700. When you call, make sure you have the
system serial number, software version number, and the system
owner’s name available.
18
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Introduction
1
• ImageXpress Velos Laser Scanning Cytometer Overview on
page 19
• How the ImageXpress Velos Laser Scanning Cytometer Operates
on page 20
• Instrument Specifications on page 22
• Connecting the ImageXpress Velos Cytometer to the Computer
on page 23
• Starting the Instrument and Software on page 26
ImageXpress Velos Laser Scanning Cytometer
Overview
The ImageXpress® Velos Laser Scanning Cytometer is a benchtop laser
scanning cytometer that enables dynamic fluorimetry, the
measurement of fluorescence emission signals and their characteristic
properties other than intensity and wavelength. The scanner’s objectbased technology measures signals associated with objects in the focal
region to:
• Distinguish object signal from background
• Quantify objects
• Measure object shape characteristics
• Measure object fluorescence intensity
• Measure object anisotropy
Note: The term object or particle refers to the finite-sized subject of
interrogation and analysis by the ImageXpress Velos Cytometer.
The cytometer’s unique optical head and four photomultiplier tubes
(PMT) enable measurement of four emission colors in the intensity
domain or two in the anisotropy domain. The cytometer is compatible
with ANSI/SBS footprint microplates of any well format, or custom
plates or microscope slides held in a frame with an ANSI/SBS footprint.
CAUTION! Before using the ImageXpress Velos Cytometer, make sure
you read and understand the safety information in the Preface on
page 9.
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19
Introduction
Note: The ImageXpress Velos Cytometer is for research purposes only.
It is not for use in diagnostic procedures.
The ImageXpress Velos Laser Scanning Cytometer includes the
ImageXpress Velos Software that enables you to:
• Acquire, view, and analyze data
• View user-specified data in the microplate map (for example,
mean, peak, or total intensity in a particular channel)
• Apply intensity or area thresholds and view particles within or
outside the limits to quickly identify particles of interest
• Perform simultaneous data acquisition and analysis
How the ImageXpress Velos
Laser Scanning Cytometer Operates
The ImageXpress Velos Cytometer focuses and scans a small laser spot
(7 μm) along the bottom of a transparent microplate, slide, flask, or
other substrate. One of two lasers is used to excite fluorophores that
are present in samples that are contained, for example, in microplate
wells. The fluorescent emission of the excited fluorophores is filtered by
user-selected emission filters, then collected and subsequently
detected by four photomultiplier tubes (PMTs).
The electrical signals produced by the PMTs are digitized by analogdigital converters at 40 MHz, producing a digital output that represents
the fluorescent intensities. The instrument detects objects that produce
signal above a user-specified background and collects dimensional
measurements of the objects for quantitation. The rate of sampling and
the sample stage movement determine the data resolution (pixel size).
As the laser moves across a microplate, the ImageXpress Velos
Software reconstructs a 2-dimensional map of object fluorescent
topography from the fluorescent intensity data. Size, shape, and
brightness define objects of interest (for example, beads or cells);
debris or other inappropriate objects are ignored.
20
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Figure 1-1 shows a schematic of the scanner optics. The scan head
consists of two optical channels with two detectors per channel. The
binocular collection optics enable you to:
• Collect up to four colors in the intensity domain, or
• Simultaneously collect up to two colors of fluorescent light
polarized parallel or perpendicular to the excitation light for
sensitive and accurate measurement of fluorescence
polarization (FP) or anisotropy.
Anisotropy measurements can distinguish between the fluorescence
from labeled molecules in solution and the fluorescence from molecules
bound to a larger molecule or bead.
Figure 1-1 Schematic of the ImageXpress Velos Cytometer laser optics
Four photomultiplier tubes enable four-color intensity measurements or
two-color anisotropy measurements.
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21
Introduction
Instrument Specifications
Figure 1-2 ImageXpress Velos Laser Scanning Cytometer
Table 1-1 ImageXpress Velos Cytometer specifications
IsoC
Item
Specification
Weight
130 lbs (59 kg)
Dimensions
(W x D x H)
19 x 31 x 15 inches (48 x 79 x 38 cm)
Laser-based excitation 405, 440, 488, 532, or 640 nm
Detectors
22
•
•
4 photomultiplier tubes (PMT)
 4 channel (color) intensity
 2 channel (color) anisotropy
Individual PMT gain adjustment
Digital Acquisition
System
•
•
•
•
40 MHz sampling rate
14 bit digitization for 16,384 discrete levels
Dual DSPs for on-the-fly processing
1GB Ethernet communication link
Sample Format
ANSI/SBS footprint microplate
Plate configuration files (.pt) for 6, 24, 48, 96, 384,
and 1536-well plates and a microscope slide
Custom plate configuration files can be specified.
Temperature
60 to 80°F
Humidity
0 to 70% non-condensing
Clearance
6 inches on each side
Mechanical stability
Free from extraneous noise and vibration
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Table 1-1 ImageXpress Velos Cytometer specifications (cont’d)
Item
Specification
Electrical supply
Either 100–120 VAC or 200–240 VAC at 50–60 Hz
Nominal Supply
Voltage
100–120 VAC (10A)
200–240 VAC (6A)
Accessory kit
Contents
• Alignment Plate(s)
• Microscope slide holder(s)
• Dichroic mirrors with holders for each laser
• Emission filters with holders for each laser
• ImageXpress Velos Laser Scanning Cytometer
User Guide
ImageXpress Velos
system computer
Specifications
• Windows operating system
• Processor: Intel multiprocessor with 2GB DRAM
• Hard drive: Minimum 500 GB
• Flat panel monitor
• ImageXpress Velos Software
Connecting the ImageXpress Velos Cytometer
to the Computer
The ImageXpress Velos Software automates control of the scanner,
image acquisition, and image analysis. The software is pre-installed on
the scanner workstation desktop.
Figure 1-3 and Figure 1-4 show the connections on the back of the
ImageXpress Velos Cytometer and the computer.
• Use the Ethernet cable to connect the ImageXpress Velos
Cytometer to the Ethernet port on the computer.
Note: The computer is configured with two Ethernet ports. Connect the
ImageXpress Velos scanner cable to the integrated ethernet port on
the computer. To add the computer to your network, connect the
network’s Ethernet cable to the port in the additional Ethernet card
located in an expansion slot. Please contact your system administrator
for assistance determining compatibility.
CAUTION! Do not reconfigure either Ethernet port or the
ImageXpress Velos Cytometer might not function.
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23
Introduction
1
2
Figure 1-3 ImageXpress Velos Laser Scanning Cytometer, rear view
Item
Description
1
Power input
2
ImageXpress Velos Cytometer Ethernet connection
Note: The user is responsible for maintaining a virus-free computer,
otherwise instrument performance and data integrity might be
diminished. Virus scans and Automatic Updates must not occur when
the ImageXpress Velos Cytometer is acquiring data, saving data, or
analyzing data.
24
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1
2
3
4
Figure 1-4 ImageXpress Velos system computer, rear view
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Item
Description
1
ImageXpress Velos Cytometer computer Ethernet connection
2
Monitor connection
3
Ethernet connection for your local network
4
Power input
25
Introduction
Starting the Instrument and Software
1. To turn on the cytometer, press the On/Off (l/0) switch located
on the rear of the instrument, above the power input.
2. Allow the ImageXpress Velos instrument to fully initialize for five
minutes before starting the software.
3. To start the ImageXpress Velos Software, double-click the
ImageXpress Velos Software icon
on the desktop.
The ImageXpress Velos Software window appears.
Figure 1-5 ImageXpress Velos Software main window
26
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Creating a Method
2
• Verify Emission Filters on page 27
• Loading a Microplate into the ImageXpress Velos Cytometer on
page 29
• Steps to Create a Method on page 31
• Using an Existing Method on page 41
A typical assay workflow includes:
1. Verify ImageXpress® Velos Cytometer hardware settings and
load the sample plate or slide. See Verify Emission Filters on
page 27 and Loading a Microplate into the ImageXpress Velos
Cytometer on page 29.
2. Open or set up a method. A new assay might require
optimization of the instrument parameters using the Calibration
screen. See Using an Existing Method on page 41 and Step 4:
Optimizing Settings in Calibration Mode on page 37.
3. Choose a process for analyzing images as they are acquired. A
new assay might require a test scan to set up the process. See
Step 4: Optimizing Settings in Calibration Mode on page 37 and
Steps to Create a Process on page 55.
Verify Emission Filters
Emission filters must be manually inserted in the ImageXpress Velos
Cytometer. The bandpass filter compartment is located at the front of
the instrument.
Several filter sets are available for use with the ImageXpress Velos
Cytometer. For more details, see Validated Fluorophores and
ImageXpress Velos Filters on page 115. When you install the filter sets,
insert the lower wavelength filters into channels 1 and 2 and place the
higher wavelength filters in channels 3 and 4.
The dichroic mirrors are typically long pass filters that allow longer
wavelength light to pass through to channels 3 and 4, and reflect
shorter wavelengths into channels 1 and 2.
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27
Creating a Method
To change emission filters:
1. Grasp the handle of the front filter compartment cover and tilt it
out and down, away from the instrument.
2. Pull the silver knob out slightly on the light-tight inner door and
slide the door to the right to expose channels 1 and 3 or to the
left to expose channels 2 and 4.
1
2
Figure 2-1 ImageXpress Velos Cytometer, emission filter
compartment
Item
Description
1
Channel 2
2
Channel 4
3. After you locate the channel that you want to change, remove
the filter by pulling it straight out.
4. Insert the new filter, mounted in a black filter holder, into the
position of interest. Slide the filter into the slot so that the white
label is facing the dichroic mirror.
5. Carefully insert the new dichroic mirror with the knurled knob at
the top. Push it in until it stops.
6. Slide the light-tight inner door closed.
7. Close the compartment cover.
28
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For Anisotropy Assays
The parallel polarization emission channels are detectors 1 and 3, and
the perpendicular polarization emission channels are detectors 2 and 4.
When you scan a plate for anisotropy, be sure to place the same
wavelength filter into the parallel and perpendicular channels (for
example, channel 1 matches channel 2 and channel 3 matches channel
4). If the method specifies polarization, the polarizing filters are
automatically moved into place.
Loading a Microplate into the ImageXpress Velos
Cytometer
Viewing the Plate Map
The plate map appears when you open a method.
Plate map toolbar buttons
Start scan
Stop scan
Load/Eject
Enlarge window
Reduce window
Figure 2-2 Plate map
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29
Creating a Method
Loading the Microplate
1. Click the Load/Eject button
in the plate map toolbar to eject
the plate holder.
2. Place the microplate in the plate holder so that the upper-left
corner of the plate (with well A1) is in the upper-left corner of
the holder.
1
Table 2-1 Plate holder
Item
Description
1
Microplate corner with well A1
3. To retract the plate holder into the scanner, click the Load/Eject
.
button
30
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Steps to Create a Method
A method is a template file that contains all of the instrument settings
that are needed to scan a plate. A unique method is required for each
type of assay to accommodate different emission wavelengths, the type
of microplate, or signal intensity for the particular experiment. After a
method is optimized for a particular assay, it can be used every time
that type of assay is run on the ImageXpress Velos Cytometer. If the
same assay will be run on a different ImageXpress Velos Cytometer,
slight adjustments to some of the method parameters, such as PMT
gain settings, might be necessary.
Before scanning a plate, you must first create a method or open a
saved method (.bbd) that specifies the following:
• Scan parameters (for example, plate type, scan area, and pixel
size)
• PMT channel(s) and channel parameters for data collection
• Wells to be scanned
• Scanner settings that optimize the signal (recommended for a
new assay)
• Process for image analysis (optional)
Note: Either a method (*.bbd) or data (*.bbd) file can be opened as a
method and all parameters will be retained for acquisition and
analysis.
Steps
•
•
•
•
•
•
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for creating a method for a new assay include:
Step 1: Set the scan parameters on page 32
Step 2: Set the detect parameters on page 33
Step 3: Set the analysis parameters on page 35
Step 4: Optimizing Settings in Calibration Mode on page 37
Step 5: Select the wells to scan on page 39
Step 6: Save the method on page 40
31
Creating a Method
Step 1: Set the scan parameters
1. Click the New button
or click File > New on the main menu.
The Instrument Setup dialog appears.
2. Click the Scan tab in the Instrument Setup dialog.
1
2
3
4
5
5
Figure 2-3 Instrument Setup dialog, Scan tab
32
Item
Description
1
Enter notes about the scan in the Description box.
2
Select a plate type from the list. The plate type specifies
information about the well dimensions and plate height.
Confirm the well area where the data will be collected
(Scan area). If you want to scan a sub-area of each well,
enter smaller dimensions.
3
Confirm the default scan resolution or select new x,y pixel
dimensions (μm). Choosing smaller numbers for scan
resolution increases the plate scan time and the size of
the saved image. See Table D-1 on page 119.
4
If you want to average multiple scans of the same pixel to
generate the image, select 2, 4, 8, or 16 from the
avgs/pixel list. This increases the plate scan time, but
does not affect image size.
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Note: The ImageXpress Velos Cytometer accommodates a standard
SBS footprint microplate of any well format. The default Plate Type list
includes many industry standard plates. If the type of microplate that
you want to scan is not in this list, you will need to create a plate
configuration file. For more details, see Creating a Plate Configuration
File on page 177.
Step 2: Set the detect parameters
1. Click the Detect tab.
2. Put a check mark next to the PMT channel(s) that you want to
use to collect signal.
Figure 2-4 Instrument Setup, Detect tab
3. For each enabled channel, confirm the default settings, or type
or select a new value for filter, polarization, gain, and sensitivity.
4. Select the laser excitation wavelength.
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33
Creating a Method
Note: It is important to optimize the scanner signal for a new assay
before scanning samples. For more details, see Step 4: Optimizing
Settings in Calibration Mode on page 37.
Table 2-2 Instrument Setup dialog, Detect tab
34
Item
Description
Filter
The emission filter wavelength color or dye description can be
entered here. You must manually insert the emission filter.
Polarization
The plane of the emission fluorescence that is collected.
• All: All emission light is collected, regardless of planar
orientation.
• Parallel: Emission parallel to the excitation light is
collected.
• Perpendicular: Emission perpendicular to the excitation
light is collected.
• Scatter: Uses a narrow band filter spanning the
wavelength of the excitation laser.
Gain
Sets the voltage of the PMT. Increasing the gain setting
increases the signal as well as the noise. Significant gain
adjustments are typically made in the Calibration window.
Sensitivity
Increase sensitivity by 2, 4, or 8 times.
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Step 3: Set the analysis parameters
If you have already created a process for image analysis (.bbp) and
want to analyze image data as it is acquired (auto-analysis), choose a
process to include in the method (click the Browse button ). For more
details on creating a process, see page 55.
1. Click the Analysis tab.
2. To include a process in the method, click Enable Image Analysis.
For more details on the analysis options, see Table 2-3.
Figure 2-5 Instrument Setup dialog, Analysis tab
Note: Real-time data analysis during scanning (auto-analysis) is
available only if the method includes a process.
To generate .csv, .iso, and .fcs files, you need to select them before
image analysis is performed. For more information on file types, see
Table B-1 on page 111.
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35
Creating a Method
Table 2-3 Instrument Setup dialog, Analysis tab
Item
Description
Enable Image Analysis
Choose this option if you have already created a
process (.bbp) for image analysis and you want to
analyze data as it is acquired (auto-analysis mode).
For more details on creating a process, see page 45.
Click the
button to select the process.
Note: If this option is selected, the software
generates a single results file (plate.txt) that
incudes the data for all scanned wells.
Sequential Analysis
Choose this option to have the system scan over all
the selected wells and acquire the data, and then go
back and analyze the wells according to the selected
process. Otherwise, the system acquires and
analyzes the data at the same time during a scan.
The benefit of Sequential Analysis is that it is less
CPU intensive.
Note: The .csv, .iso, and .fcs file formats are available only if you
choose the Enable Image Analysis option. For more information on file
types, see Table B-1 on page 111.
36
Produce individual .csv
files
Choose this option if you want to produce an
individual results file .csv (comma-separated
variable) for each well.
Produce individual .iso
files
Choose this option to generate files in Flow
Cytometry Standard Express format.
Produce individual .fcs
files
Choose this option to generate files in Flow
Cytometry Standard format.
Produce data (.bbd) file
Choose this option to save the images and data. If
you are reanalyzing a previously scanned plate, you
might not want to choose this option to avoid
resaving the plate image file. If you save the .bbd,
you can use this file to generate other files or data
formats.
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Table 2-3 Instrument Setup dialog, Analysis tab (cont’d)
Item
Description
Post Process Name
Choose this option if you want to apply a post
process (.ppr) to the image data. The post process
(for example, Toxcount Live/Dead cell assay or
Mitotic Index assay) summarizes the data in a text
file (.txt). Click the
button to select the post
process file. Raw data is typically analyzed in
another program, such as an Excel macro, to
determine the correct slope and intercept to classify
the points on a scatter plot. The slope and intercept
can be entered under the post-process in the Ratio
and Intercept boxes and used for analysis.
If this option is selected, the data analysis is specific
to the assay type and a second file is generated in
the plate.txt folder.
Note: This option is available only if you
choose the Enable Image Analysis option.
Produce MDCStore™
Compatible files
Generates files that are compatible with the
MDCStore database (one file per channel acquired
per well). Files in this format can be analyzed using
the MetaXpress® Software.
Auto. Correct Saturated Corrects saturated data points, which can be zero, in
Data
an image using values from the surrounding areas
for compatibility with MetaXpress Software or other
image analysis programs.
Step 4: Optimizing Settings in Calibration Mode
It is important to optimize the scanner signal for a new assay. The goal
of optimization is to make adjustments so that the:
• Amplitudes of the signals of interest are about 50% to 75% of
the vertical scale in the Scope window.
• Signal is balanced across the detector channels so that the
amplitude is nearly the same across the enabled detector
channels.
Note: A balanced signal is not required for data acquisition, but it is
useful for making anisotropy measurements where the calculations
use signals from the perpendicular and parallel polarization channels.
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37
Creating a Method
To optimize the signal, you can adjust the following parameters:
• PMT gain and sensitivity of a detector channel
• Collection focus (the distance between the collection optics and
the microplate)
• Scanning focus (distance between the laser scanning lens and
the microplate)
Steps to optimize the scanner signal include:
• Step 1: Open a method and load a plate with samples on
page 41
• Step 2: Open the calibration screen and view the real-time
signal on page 42
• Step 3: Select the detector channel(s) and plate column or well
to display in the scope window on page 44
• Step 4: Adjust the scan line position to find the best objects or
region to focus on on page 46
• Step 5: Adjust the collection or scanning focus on page 48
• Step 6: Adjust the signal amplitude on page 51
• Step 7: Save the optimized parameter settings with the method
on page 52
Note: Photobleaching can occur in the wells used for signal
optimization. Therefore, it is recommended that you prepare control
wells specifically for the optimization process. This can be done in a
separate plate of the same type used for the assay or you can use a
single column of the assay plate. If you are optimizing the signal for an
anisotropy assay, make sure that the plate includes wells with
unbound labeled tracer and use these wells to optimize the signal.
Balance the signal for anisotropy measurements without the
anisotropy filters chosen in Instrument Setup.
38
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Step 5: Select the wells to scan
1. Click OK after you finish setting the parameters in the Scan,
Detect, and Analysis tabs.
Figure 2-6 Select wells to scan
2. In the plate map that appears, left-click and drag the mouse to
select around the wells that you want to scan.
The wells selected for scanning are outlined in blue.
3. To deselect a wells, right-click and drag the mouse to deselect
the wells.
Note: When you select wells, the selection box moves to the
farthest left position of the mouse.
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39
Creating a Method
Step 6: Save the method
1. Click the Save Method As button
. Alternately, click File > Save
Method As on the menu bar.
2. In the dialog that appears, confirm the directory where the
method will be saved or select another directory, name the
method, and click Save.
Figure 2-7 Saving a method
Note: You must save a method again after you optimize the scanner
signal from a new assay so that the method includes all of the
optimized parameter settings. For more details, see Step 7: Save the
optimized parameter settings with the method on page 52.
40
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Using an Existing Method
Step 1: Open a method and load a plate with
samples
1. Confirm that the correct laser is enabled and the correct
emission filters are inserted into the scanner.
2. Load a sample-filled microplate in the cytometer. See Loading a
Microplate into the ImageXpress Velos Cytometer on page 29.
3. Click the Open Method button
or click File >Open Method on
the menu bar.
4. In the dialog that appears, select the method of interest and
click Open.
Figure 2-8 Opening a method
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41
Creating a Method
Step 2: Open the calibration screen and view
the real-time signal
The scope window displays a real-time signal that enables you to
observe how adjustments to the PMT gain or focus settings affect the
signal. In the calibration control panel, you can specify the detector
channel(s) and the row or well to display in the Scope window, adjust
instrument focus, and optimize gain settings for each PMT.
1. Click Calibrate > Calibration on the menu bar.
The calibration screen appears.
2. In the Go to Column field, type a column number from the plate
that contains a positive signal.
to display the real-time signal.
3. Click the Start button
4. If necessary, change the PMT gains to adjust signal peaks to
between 50% and 75% of the window height (Figure 2-9).
5. Close the Calibration window: Click Apply n Quit.
6. Scan the plate and save the data.
42
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1
2
6
3
4
5
Figure 2-9 Scope window
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Item
Description
1
Scan across one column
2
PMT signal from one bead or cell
3
Approximate well locations (96-well plate)
4
Start of scan (row A)
5
End of scan (row H)
6
Calibration control panel
43
Creating a Method
Step 3: Select the detector channel(s) and
plate column or well to display in the scope window
1
2
3
5
4
Figure 2-10 Calibration Control Panel
44
Item
Description
1
Select one or more detector channels to display in the Scope
window.
2
To view the signal from a particular column (Figure 2-11), enter a
column number in the Go to column box and click Set. For
example, if you want to view column 2, enter “2” in the box.
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Figure 2-10 Calibration Control Panel
Item
Description
3
To view the signal from a single well (Figure 2-12):
 Enter a row letter in the View Well box and click Set.
 Enter a column number in the Go to column box.
For example, to view well A3, enter 3 in the Go to Column box
and A in the View Well box.
4
To adjust the area of the well displayed in the Calibration screen,
change the x-axis by entering a measurement in the Width of
(μm) box and click Set.
5
Sets the scale of the x-axis in the Scope window.
Note: If there is no column entry, the Scope window displays the signal
from the current position of the laser, wherever that might be. If the
plate was just loaded into the instrument, the laser might not be in a
position to scan any plate wells. As a result, all of the signals visible in
the Scope window will be close to baseline.
Figure 2-11 Scope window, entire column displayed
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45
Creating a Method
Figure 2-12 Scope window, a small area of a single well displayed
Note: If the signal is very low, it might mean that the laser focus and
collection optics are not located in a region that has sufficient objects
or signal of interest. Use the Scan Line adjustments to move the scan
line around within the column to find fluorescent objects.
Step 4: Adjust the scan line position to find the
best objects or region to focus on
1. Confirm the default or enter a new value for the distance that
the scan line is moved (left or right) when you click the
arrows.
2. To move the scan line left or right, click the or arrow.
The change in position (Delta) and the current location
(Position) are updated in the control panel.
46
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1
2
3
Figure 2-13 Calibration control panel
Item
Description
1
Current position of the scan line (to the right of the left plate
edge)
2
Change in the scan line position from the position specified by the
plate type
3
Incremental distance that the scan line moves when you click the
arrows
Note: During signal optimization, be aware of the length of time the
scan line is located at a particular well or column. If you optimize the
signal at a photo-bleached location, the signal can be much larger at
other scan locations when the plate is scanned.
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47
Creating a Method
Step 5: Adjust the collection or scanning focus
The value of the collection focus is the distance between the collection
optics and the home position of the collection optics. Increasing this
value moves the lens closer to the bottom of the plate and moves the
focus position up into the well. The scanning focus is the distance
between the scanning lens and the home position of the scanning lens.
Increasing this value moves the laser focus up into the well.
The optimum focus settings maximize the signal amplitude and
sharpness (at a particular gain and sensitivity setting). You can move
the collection optics or the laser focus up or down in user-modifiable
steps and observe how the signal changes in the Scope window. The
software provides a default collection and scanning focus setting. Small
incremental changes can be made to optimize the amplitude and
sharpness. The depth of focus is ±200 μm and allows a large region of
the bottom of the well to be in focus.
Note: When adjusting the collection focus, make sure to adjust the
scanning focus by the same increment. For example, if you move the
collection focus up by 500 μm, move the scanning focus up by
500 μm. For more details on viewing a single well, see Step 3: Select
the detector channel(s) and plate column or well to display in the
scope window on page 44. If necessary, click the Adjust view arrows
to scroll the view in the Scope window.
To change the position of the collection optics:
1. Confirm the distance of the step change in the Collection Focus
box (default = 100 μm) or enter a new value  1000 μm.
2. Click the Collection Focus up or down arrow
48
.
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1
2
3
4
Figure 2-14 Calibration control panel, changing the position of
the collection optics
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Item
Description
1
Scan Line: The user-specified distance (μm) that the
collection optics are moved up or down when you click the
or
button.
Delta (μm): The distance the collection optics have been
moved (up or down) from the original position.
Position (μm): The current position of the collection optics
above the home position.
2
Incremental change in position that is applied when you
click the
arrows.
3
Change from the original position of the collection or
scanning focus.
4
Click to transfer modified collection and scanning focus
settings to a method before closing the Calibration
window.
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Creating a Method
Note: The confined detection region of 400 μm allows
extraneous background fluorescence to be ignored.
3. After adjusting the focus, click Reset to display the entire column
so you can confirm that the signals are optimized with respect to
amplitude and balance across the entire column.
4. Click Update Method Home to transfer any changes made to the
focus to the current method.
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Step 6: Adjust the signal amplitude
The signal amplitude can be increased or decreased by adjusting the
gain settings of each photomultiplier tube (PMT) (the detectors).
If multiple PMT detector channels are enabled in the method, you must
optimize the signal amplitude of each one. In anisotropy assays, it is
important that the paired channels (1 and 2 or 3 and 4) are balanced.
1. Select the channel(s) to display in the Scope window.
2. Select the channel that you want to adjust.
3. To increase the signal amplitude, enter a higher value in the PMT
Gain box and click Set. You can also select a higher Sensitivity
factor.
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Creating a Method
The sensitivity factor multiplies or amplifies the signal that is
provided by the PMT. A higher sensitivity setting amplifies both
the signal and the background.
4. To decrease the signal amplitude, enter a lower value in the PMT
Gain box and click Set. Generally, a 1X sensitivity (no correction)
is adequate.
Table 2-4 Calibration control panel, Channel adjustments
Item
Description
Channel
Selects the detector channel that you want to adjust.
PMT Gain
Specifies a value for the gain (voltage across the PMT).
Increasing the gain, increases the signal amplitude. The PMT
gain can be set between 0 to 1200 mV. The limits of stability for
control of the gain are 200 to 900 mV. Outside these limits, the
PMT operates in an unstable region. Since the control of the
PMT gain is most stable between 200 and 900 mV, it is
recommended that you choose a gain setting within this range
when calibrating the instrument.
To change the gain setting, start by adjusting the voltage in
increments of 20 and click Set.
Sensitivity
Specifies a multiplier for the preamplifier that amplifies the
signal from the PMT.
Step 7: Save the optimized parameter settings
with the method
The optimized parameters that are saved with the method include the
scan focus position, collection focus position, PMT gain for each
channel, and the sensitivity setting for each channel.
1. After you finish optimizing the signal, click Update Method Home
to save the changes to the current method.
Note: The Update Method command applies the changes made
to the Collection Focus and the Scanning Focus only. If you are
just optimizing the PMT gains and sensitivity, the changes
automatically update in the Method.
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Figure 2-17 Scope window
2. To exit the calibration process click Calibration > Calibration on
the menu bar or click Apply to close the Calibration window.
3. Click the Save Method As button
details, see page 40.
to save the method. For more
Note: If the method will be used in a robotic protocol, be sure to also
select the wells to be scanned in the plate map before you save the
method. Otherwise, no wells will be read from the method.
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Creating a Method
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Creating a Process
3
• Steps to Create a Process on page 55
• Viewing Processed Well Data on page 75
• Viewing the Particle Summary on page 78
• Customizing the Well Results on page 80
• Saving Analysis Results on page 81
• Manually Analyzing Data on page 81
The ImageXpress® Velos Software provides image and particle (object)
analysis tools. A process (.bbp) specifies the parameter settings that
are used to identify objects in the image. After you save a process,
update your method to include the process so that the method can
analyze data in real time during scanning (auto-analysis).
Steps to Create a Process
The steps to create a process include:
• Step 1: Open image data (.bbd) on page 56.
• Step 2: Specify a region of interest (ROI) (optional) on page 57.
• Step 3: Set the brightness and contrast on page 59
• Step 4: Open the analysis processing control panel on page 61.
• Step 5: Optimizing the intensity threshold on page 62.
• Step 6: Choose any additional processing and anisotropy factors
on page 65
• Step 7: Set the particle filters on page 67.
• Step 8: Test and optimize the process on page 68.
• Step 9: Save the process on page 74
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Creating a Process
Step 1: Open image data (.bbd)
1. Open the image data (.bbd) (click the Open button
or click
File > Open Data on the menu bar).
2. Double-click a well in the plate map.
The image window appears. The entire window can be resized
by clicking and dragging the image edges to the desired size.
Note: Multiple wells can be processed at the same time.
Table 3-1 Image window toolbar
Item
Description
Clicking this button changes the mouse arrow to a
. In
this mode, you can grab and move the image within the
window (press and hold the mouse button while you move
the mouse).
Click this button and then click the image to magnify the
image. Double-clicking on the image will return the image
in the window to its original size.
Click this button to change the mode to Select.
Click this button and then click the image to zoom out on
the image.
Opens the Brightness and Contrast dialog.
Opens the Region of Interest dialog.
Click a channel to view the scan image from that channel.
Click this button to change the colors for Channels 5
and/or 6.
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Step 2: Specify a region of interest (ROI)
(optional)
If you specify an ROI on an image, the software ignores objects outside
the ROI and excludes them from analysis.
button on the image window
1. To draw an ROI, click the
toolbar or click Image > Region of Interest (ROI) on the menu bar.
A yellow handle
appears at the center of the image and the
Region of Interest dialog appears.
2. In the dialog, select the Circular or Rectangular option.
Note: If a yellow line denoting the ROI does not appear on the image,
it might be larger than the scanned image. In the Region of Interest
dialog (Figure 3-1), click Default to display a starting ROI that is
appropriate for the well size.
Figure 3-1 Drawing an ROI on an image
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Creating a Process
Note: The numbers that are indicated in the image are the actual
dimensions of each pixel in the image. The values correspond to a
calibrated measurement that is made during instrument manufacture
that relates the size of the pixel to the dimension of a real object. Each
system that is built has a slightly different calibration, so the actual
size of the pixel will vary from system to system. The software does
not account for the real calibration; it simply calls out a nominal pixel
size, such as 5 μm x 5 μm.
3. Put the mouse arrow over the yellow handle
at the center of
the image. When the double arrow
appears, press and hold
the mouse button while you drag the handle to draw the ROI.
As you draw the ROI, its location and dimensions are
automatically updated in the Region of Interest dialog.
4. To change the ROI size, drag a handle . Alternately, enter new
diameter for a circular ROI or a new width and height for a
rectangular ROI.
Figure 3-2 Changing the ROI size
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5. To move the ROI, put the mouse arrow anywhere inside the
ROI, and then press and hold the mouse button while you move
the mouse arrow. Alternately, enter new values for the X center
offset and Y center offset in the dialog.
Figure 3-3 Moving an ROI
6. To set the ROI, run the process.
Note: The specified ROI is saved with the Process.
Step 3: Set the brightness and contrast
Note: When an image is initially opened before it is processed, the
brightness and contrast settings from the previously opened image are
applied.
1. Click the
button on the image window. Alternately, click
Image > Brightness and Contrast on the menu bar.
2. In the dialog that appears, use the sliders to adjust the image
settings (for more details see Table 3-4).
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Creating a Process
Intensity (x-axis) vs. number of pixels (y-axis)
Figure 3-4 Image brightness and contrast controls
Item
Description
Brightness control.
Contrast control.
Non-linear contrast correction of the image.
Adjusts intensity of different object colors.
Default
Restores all settings to the defaults.
Apply to All
Applies the settings to all channels in all wells of the plate.
The settings are applied to the channel selected from the list
unless you select Apply to All. The intensity histogram shows
the results for the selected channel.
Enables you to designate a color for each channel (gray, blue,
green, or red).
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Step 4: Open the analysis processing control panel
•
Click the Process toolbar button
.
Alternately, open a well image (double-click a well in the plate
map), then click Analysis > Process on the menu bar.
Table 3-2 explains the items in the analysis processing control
panel.
Analysis processing control
Figure 3-5 Opening the analysis processing control panel
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Creating a Process
Step 5: Optimizing the intensity threshold
The software ignores pixels within a designated bottom and top range.
The bottom threshold intensity helps distinguish signal from
background noise.
You can set a fixed threshold that is the same for all wells or an
adaptive threshold that varies by well. For an adaptive threshold, the
software uses the baseline% value to compute the background signal
on a per well basis. For example, if baseline% = 10%, the software
identifies 10% of the total pixels with the lowest intensity and
computes their average intensity. The software adds this average
intensity to the user-specified offset to generate the bottom threshold.
Figure 3-6 Intensity threshold components. A histogram of pixel intensities
in one image generated from a microplate well. The blue plot shows that the
largest number of pixels have low intensity, and are background signal. The
smaller population of higher intensity pixels represent the bright objects in the
image.
A fixed threshold can be optimum for homogeneous biochemical
assays. The adaptive threshold and a baseline% in the range from 5%
to 20% can be optimum for bead or cell-based assays. It is
recommended that you start with the baseline% set to 10%.
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To set a fixed intensity threshold:
1. Set the baseline% = 0.
2. Enter a value for the offset and Max intensity threshold.
Figure 3-7 Analysis processing control panel
3. Select a channel or combination of channels from the Detect
particles in list.
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Creating a Process
To set an adaptive intensity threshold that varies by well:
1. Enter a value for the Baseline%.
2. Enter a value for the offset and max intensity threshold.
3. Select a channel or combination of channels from the Detect
particles in list.
Table 3-2 Analysis processing control panel, Threshold parameters
Item
Description
Baseline%
A factor used to compute background that is added to the
lower limit of the user-specified intensity threshold to
generate an adaptive threshold. It is recommended that
you start with a value of 10%. This means that the
software identifies 10% of the total pixels with the lowest
intensity, then computes their average intensity. The
computed value is added to the user-specified lower
threshold to determine the adaptive threshold.
Offset
The lower limit of the intensity threshold that distinguishes
signal from background. Objects with an intensity less
than the bottom threshold are removed from the analysis.
It is recommended that you start with a bottom value of
25%.
Max
The upper limit of the intensity threshold. Objects with an
intensity greater than the top threshold are removed from
the analysis. The highest possible intensity value is 65535
rfu.
Detect particles in Select the channel or combination of channels that you
want to use to identify the objects.
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Step 6: Choose any additional processing and
anisotropy factors
Note: For more details on the additional processing items, see page
Table 3-2 on page 64.
Figure 3-8 Additional processing and anisotropy settings
•
•
•
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Formula: A list of corrective calculations that can be applied to
the image data. If you do not want to apply a corrective
calculation, select None.
Smoothing: A method to help remove noise from the data. To
apply smoothing, enter a number from 1 to 5. If you do not
want to apply smoothing, enter zero in the Smoothing box.
Typically smoothing of 0 to 2 is used for cell-based assays.
Flattening: A method to help reduce background and improve
thresholding. To apply flattening, enter a number from 1 to 3000
in the Flattening box. If you do not want to apply background
flattening, enter zero in the box. Be sure the number you enter
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Creating a Process
•
66
for flattening is larger than the size of the objects you want to
identify. Typically flattening is set between 100 μm and 300 μm.
Anisotropy factors: For anisotropy assays, a g-factor corrects for
biases in polarization due to interference from optical elements.
If you do not want to apply a correction, enter 1.00 in the g1 or
g2 box.
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Step 7: Set the particle filters
Particle filters have three conditions:
• Excluded: Excludes objects that are outside the set range.
• Included: Includes objects that are inside the set range.
• Do not use: The filter is not applied.
The area filter is useful for ignoring contamination (for example, large
fibers or very small particles) or discriminating between individual and
aggregated cells. To filter objects by size, use the area filter to specify
the area of the object that you want to process. For 10 μm beads or
cells, set the Min to 100 μm and Max to 800 μm.
1. To set a particle filter, enter the minimum and maximum value.
Figure 3-9 Particle filter settings
2. Make a selection from the Condition list.
3. Verify and optimize each filter parameter.
4. Save the process.
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Creating a Process
Step 8: Test and optimize the process
After you specify the process parameters and other analysis settings,
test the process on an image.
1. Open the image(s). Multiple images can be processed at the
same time.
Figure 3-10 Open images for processing
2. Open the Analysis processing control panel (click the Process
toolbar button
).
3. Click the Browse button
and in the dialog that appears, select
a temporary directory for the results generated during the
testing of the Process (.csv). If no directory is specified, all the
results files will be saved onto your desktop.
Note: This CSV directory is not the folder where final data are saved
after analyzing and saving the plate data.
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A log of the processing
steps during the analysis.
Figure 3-11 Selecting a temporary directory for the results
4. Click Process!.
The software analyzes and updates the image, and then displays
the well results (.csv). The .csv format includes information
about each object in the well. For more details on the well
results, see Table 3-3.
Figure 3-12 Well results (.csv)
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Creating a Process
5. Check the processed image and the well results (.csv) to
determine if the process generates expected results. Do any of
the following:
 Select (by clicking) one or more rows in the results.
 The selected particles are outlined in red (or other specified
color) in the image.



Click the button in the image window toolbar, then click a
particle in the image.
The particle is outlined in red (or other specified color) and
the corresponding row is highlighted in the results.
Press Ctrl + A keys to select all rows and highlight all objects
that pass the threshold and filter criteria.
Note: The data can be sorted by column simply by clicking on
the column header. Each time you click, it switches between
ascending and descending order.
Figure 3-13 Processed image and well results
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Note: For details on how to customize the well results table, see
Customizing the Well Results on page 80.
Table 3-3 Well results (.csv)
Item
Description
PID
Particle identification number
X
x-coordinate of the particle [μm]
Y
y-coordinate of the particle [μm]
AREA
Particle area [μm2]
AREA-PIX
The number of pixels in the particle.
SHAPE FACTOR
Ratio of object perimeter to area
ANI-1
Calculated anisotropy (channel 1 and
2)
ANI-2
Calculated anisotropy (channel 3 and
4)
IMEAN-1
Average pixel intensity in the particle.
Note: The number (1, 2, 3, or 4) that
follows the data name indicates the
channel. The number 5 or 6 indicates
data generated after a formula was
applied during processing.
IPEAK-1
Peak pixel intensity in the particle.
IINT-1
Total integrated pixel intensity in the
particle.
IBGND-1
Average background intensity in the
entire region of interest.
6. If the process allows too much background or dim objects,
increase the threshold Offset value.
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Creating a Process
Figure 3-14 Offset setting
7. To reduce background and improve thresholding, flattening is
often set to 150.
 If the objects are smaller, decrease flattening to 100
 For clumps of cells or colonies, increase flattening to 1500
 For confluent cells or solutions, set flattening to 0 (turns off
flattening)
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Figure 3-15 Flattening setting and area filter
8. If you are analyzing a total well response that includes all cells,
but you want to omit small debris, set the Area particle filter to:
 Min = 0
 Max = 100
 Condition: “Excluded”
9. If you are analyzing single cells, set the Area particle filter to:
 Min = 100
 Max = 1000
 Condition: “Included”
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Creating a Process
Step 9: Save the process
1. Click Save.
Figure 3-16 Saving a process
2. In the dialog that appears, enter a name for the process (.bbp)
and click Save.
Note: After you save the process, include it in a method so that image
data can be analyzed in real time during scanning (auto-analysis) or
use the process to re-analyze data already scanned. For more details
on how to include a process in a method, see Operating in Autoanalysis Mode on page 91.
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Viewing Processed Well Data
1. Click the
button (this puts the software in analysis mode).
2. Double-click a well.
The image window appears and shows the processed data.
Figure 3-17 Processed well data
3. Click the
button.
4. In the Analysis processing control panel that appears
(Figure 3-18):
 Click Load and select a process.
Click the Browse button
and select a directory for the
output (.csv).
 Click Process.
The identified particles are highlighted in the image and the well
results (.csv) appear.

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Creating a Process
5. If you want to view processed data for other wells, repeat step
step 2 to step 4.
Figure 3-19 View a particle tooltip or highlight a particle in the
image or table
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6. Put the mouse pointer over a particle in the image to view a
tooltip that shows particle information. If you click a particle in
the image, the corresponding row in the well results is
highlighted. If you select one or more rows in the wells results,
the corresponding particles are highlighted in the image.
7. To customize the highlighted particle colors in the image:
 Right-click the image and select Options on the shortcut
menu.
 In the dialog that appears, click Particle to choose the color
that is used to outline all particles identified by the process.
 Click Selected Particle to choose the color that is used to
highlight particles that you select in the well results.
Put a check mark next
to Show Text Info to
show well image
information.
Figure 3-20 Particle display options
8. You can save the open well image(s) and results data (.csv
file(s)) together as an analysis result (.bba):
 Click File > Save Analysis Results on the menu bar.
 In the dialog that appears, select a folder for the results, and
click Save.
The well image and the results table appear.
Note: It might be helpful to save analysis results for a control
well to provide a useful reference for future assays.
9. To close the analysis results, close the image window.
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Creating a Process
10. To view analysis results:
 Click File > Load Analysis Results on the menu bar.
 In the dialog that appears, select a .bba file, and click Open.
Viewing the Particle Summary
1. Open one or more well images.
2. Click Analysis > Summary on the menu bar.
The particle summary information for the well appears.The
particle summary includes averages for the well and total values
for the well.
Figure 3-21 Particle summary information
Table 3-4 Particle summary, well averages
78
Item
Description
X
The average x-coordinate of the particles [μm]
Y
The average y-coordinate of the particles [μm]
AREA
The average particle area [μm2]
AREA-PIX
The average number of pixels in a particle.
SHAPE
FACTOR
The average shape factor of the particles.
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Table 3-4 Particle summary, well averages (cont’d)
Item
Description
ANI-1
The average calculated anisotropy (channel 1 and 2)
ANI-2
The average calculated anisotropy (channel 3 and 4)
IMEAN-1
Average pixel intensity in all of the particles. Note: The number
(1, 2, 3, or 4) that follows the data name indicates the channel.
The number 5 or 6 indicates data generated after a formula was
IPEAK-1
Average peak pixel intensity in the particles.
IINT-1
Average total integrated pixel intensity in the particles.
IBGND-1
Average background intensity in the entire image.
Table 3-5 Particle summary, well totals
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Item
Description
X
The sum of the x-coordinate of all particles.
Y
The sum of the y-coordinate of all particles.
AREA
The total area in all particles [μm2]
AREA-PIX
The total number of pixels in all particles.
ANI-1
The total calculated anisotropy (channel 1 and 2)
ANI-2
The total calculated anisotropy (channel 3 and 4)
IMEAN-1
Total pixel intensity in all of the particles. Note: The number (1,
2, 3, or 4) that follows the data name indicates the channel. The
number 5 or 6 indicates data generated after a formula was
IPEAK-1
Summation of the peak pixel intensity in each particle.
IINT-1
Total integrated pixel intensity in the particles.
IBGND-1
Total background intensity in the entire image.
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Creating a Process
Customizing the Well Results
You can choose the types of results to include in the well results (.csv)
or the particle summary. (For more details on the particle summary,
see Viewing the Particle Summary on page 78)
1. Open an image.
2. Click Analysis > Columns on the menu bar.
Figure 3-22 Particle Columns dialog
3. The dialog that appears shows the column headers currently
selected for the well results.
 To exclude a column header from the well results:
 Click an item in the Selected box.
4. Click the
button.
The item appears in the Available box.
5. To reorder the columns in the well results, choose an item in the
Selected box, then click Up or Down.
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Saving Analysis Results
1. To save the open well image(s) and results data (.csv file(s))
together as an analysis result (.bba):
 Click File > Save Analysis Results on the menu bar.
 In the dialog that appears, select a folder for the results, and
click Save.
Note: It might be helpful to save analysis results for a control
well to provide a useful reference for future assays
2. To close the analysis results, close the image window.
3. To view analysis results:
 Click File > Load Analysis Results on the menu bar.
 In the dialog that appears, select a .bba file, and click Open.
Manually Analyzing Data
You can analyze all of the well images in the plate or a single well
image.
To analyze a single well:
1. Open the image data (.bbd) (click the Open button
File > Open Data on the menu bar).
2. Double-click the well of interest in the plate map.
The well image is displayed.
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or click
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Creating a Process
Figure 3-23 Well image
3. Click the Process button
.
The Analysis processing control panel appears.
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4. In the Analysis processing control panel, click Load and select a
process.
5. Confirm the CSV directory for the analysis results or click the
to select a different folder.
Browse button
6. Click Process.
The identified particles are highlighted in the image and the well
results (.csv) appear.
Well
Figure 3-25 Well results
Note: If you click a particle in the image, the corresponding row in the
well results is highlighted. If you select a one or more rows in the wells
results, the corresponding particles are highlighted in the image.
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Creating a Process
To analyze multiple wells or an entire plate of previously
scanned images:
1.
2.
3.
4.
5.
Open the image data (.bbd).
Under Instrument Setup, open the Analysis tab.
Check to Enable Image Analysis and browse for Process name.
Click OK.
Wells in the plate map that are outlined in blue will be
reanalyzed. If you want to analyze different wells, use the
mouse to select the wells.
6. Under Data drop-down menu, choose Analyze Data.
7. After the analysis is complete, go to the File menu and select
Save Data As.
8. Name the folder to save the results.
Note: Reanalysis of processed images can also be completed in this
way if the original process did not identify the objects satisfactorily.
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Acquire and Save Images
4
• Scanning a Plate and Viewing Images on page 86
• Saving Image Data on page 88
• Discarding Data on page 89
• Exporting Data on page 90
The ImageXpress® Velos Software controls the ImageXpress Velos
Cytometer. This chapter explains how to scan a plate and acquire
image data. For details on how to analyze images, see Creating a
Process on page 55.
Note: If you plan to operate the scanner with the Twister® II
Microplate Handler (a robotic plate mover) or a simulated robotic plate
handler (for kinetic assays), set up the autorun first, and then begin
the image acquisition workflow. For more details on working in autorun
mode, see Working in Autorun Mode on page 121.
Image acquisition steps include:
1. Open a method (.bbd). See Using an Existing Method on
page 41.
A method specifies the scanning parameters. See Creating a
Method on page 27.
Note: If the method includes a process, the software processes
(analyzes) the image data as they are acquired. See Creating a
Process on page 55.
2. Insert the appropriate emission or polarization filters into the
scanner. See Verify Emission Filters on page 27.
3. Start the autorun or load a plate into the scanner and start the
scan. See Loading a Microplate into the ImageXpress Velos
Cytometer on page 29.
Acquires the image data.
4. If not operating in auto-analysis mode, save the data for
processing at a later time. See Saving Image Data on page 88.
5. In auto-analysis mode, the image data (.bbd) and analysis
results (.csv and .plate.txt) are automatically saved.
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Scanning a Plate and Viewing Images
After you create a method (see Creating a Method on page 27) and
optimize the scanner signal (recommended for a new assay), you are
ready to scan a plate.
Starting a Scan
1. Open a method and in the ImageXpress Velos Software, verify
that the emission filters are correct for the assay and the
method.
2. Load the microplate. Click the
button in the plate map to
retract the plate holder into the scanner.
3. To start the scan, click the Start button
in the plate map.
4. In the plate map, the well appearance indicates:
A well selected for scanning
The well has been scanned, but the data have not been
analyzed.
Note: If no wells are selected, the scan will not start.
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5. To view a well image (raw data), double-click a well.
Image window (raw data)
Figure 4-1 Viewing raw data
6. To magnify or reduce the image, click zoom buttons (
the analysis viewer toolbar.
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) on
87
Saving Image Data
You must manually save the image data (.bbd), otherwise, the data are
discarded when you close the method.
Note: If you are using auto-run, images and data are saved
automatically.
To save image data:
1. After the scan is completed, click the Save As button
.
Alternately, click File > Save Data As on the menu bar.
2. In the dialog that appears, select a folder for the data, enter a
file name, and click Save.
Figure 4-2 Saving image data
It is recommended that you re-save the method immediately after you
save the data. To save the method, click File > Save Method As on the
menu bar or click the
button. The method is saved independently
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from the data and includes the “Instrument Setup” information (Method
derived from ___.bbd).
Note: Save the data first, then save the method. If you save the
method first, the data are discarded.
To open image data:
1. Click the Open button
.
2. In the dialog that appears, select the data (.bbd) and click Open.
Discarding Data
The image data can be discarded either before or after a Save. This
enables you to rescan the plate if the acquisition settings were not
correct or to erase images from a data file and re-scan using the exact
same original settings.
1. Click the Open button
.
2. In the dialog that appears, select the data file (.bbd) and click
Open.
The plate map appears.
Figure 4-3 Plate map with image data
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89
3. Click Data > Discard Data on the menu bar.
The data are deleted.
Figure 4-4 Plate map, image data deleted
Exporting Data
You can export image data to a graphic file.
Exporting Multiple Graphic Files
You can export all selected wells of data as .tif files for analysis in thirdparty software.
1. Select scanned wells to export by highlighting them in the plate
map.
2. From the Data menu, click Export Tiff Images.
3. Select a folder for the saved images.
90
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5
Viewing Data Files
Topics covered in this section:
• Operating in Auto-analysis Mode on page 91
• Viewing Results on page 93
• Saving the Image Data on page 95
If the method includes a process, the image data are analyzed in real
time during scanning (auto-analysis mode). If you do not operate in
auto-analysis mode, you can analyze the data after the scan is finished.
Operating in Auto-analysis Mode
1. Open the method (.bbd) that you want to use (click the Open
Method button
).
2. Click the Instrument Setup button
.
Figure 5-1 Open a method (.bbd)
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91
Viewing Data Files
3. In the dialog that appears, click the Analysis tab. (For more
details on the items in the Analysis tab, see Table 3-2 on
page 64.)
Figure 5-2 Instrument Setup dialog
4. Choose the Enable Image Analysis option.
5. Click the Browse button
to select a process.
6. If you want to apply a post process:
 Click the Browse button and select the post process.
 Put a check mark next to Post Process.
Note: Post processes are assay specific.

Enter a Ratio and Intercept value.
Note: The method does not save the ratio and y-intercept
values. Manually enter the values after you open the method.
92
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7. Click OK.
8. Confirm that the correct emission and/or polarization filters are
in the scanner, load the plate, and start the scan.
When the scan is finished, save the data. For more details, see
page 95.
After scanning and analysis, the plate map shows a heat map of
the highest (red) to lowest (green) number applied across the
plate.
data is not analyzed
to
analyzed wells that
are color-coded to
indicate the well with
the lowest and
highest result value
Figure 5-3 Plate map after scanning and analysis
Viewing Results
In the plate map, analyzed wells are color-coded like a heat map
according to a user-selected result measurement, for example, the
number of particles in a well. The example in Figure 5-4 shows a plate
map after analysis. The number of objects per well is the result
selected for display.
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93
Viewing Data Files
= maximum result
= minimum result
Put the mouse pointer over a well to
view a tooltip that shows the well
The wells with the minimum
and maximum results
Figure 5-4 Plate map, analyzed wells
Changing the Result Displayed in the Plate Map
1. Right-click a well in the plate map and click Select Default Result
on the shortcut menu that appears.
Figure 5-5 Plate map, shortcut menu
2. Make a selection on the shortcut menu.
The plate map is updated.
94
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Saving the Image Data
You must manually save the image data (.bbd). When you save the
data, the software generates the analysis results. See Table 5-1.
The method is saved independently from the data and includes the
“Instrument Setup” (Method derived from ___.bbd).
CAUTION! Save the data before you save the method. Otherwise, the
data will be discarded. If you close the method without saving the data,
the data are discarded.
Table 5-1 Analysis results
File
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Description
See
Page
plate.txt Summarizes the results data across all of the scanned
wells in the plate.
97
well.csv
Provides data on the particles in a particular well. One
.csv is generated per well if you selected Produce
individual csv files in the Instrument Setup dialog (see
page 36).
99
.bbd
The image data. The .bbd is generated if you selected
Produce data (.bbd) file in the Instrument Setup dialog.
35
.fcs
The Flow Cytometry Standard file format. Files in this
format can be viewed using third-party software (for
example, FlowJo, FCS Express). One .fcs file is
generated per well if you selected Produce individual fcs
files in the Instrument Setup dialog.
35
.tif
A graphic image file that MetaXpress or MetaMorph
Software can import.
35
.iso
A file format compatible with FCS Express. Contains
both image and data.
35
95
Viewing Data Files
To save processed data:
1. After the scan is completed, click the Save As button
Alternately, click File > Save Data As on the menu bar.
.
Figure 5-6 Saving processed data
2. In the dialog that appears, select a folder for the data, enter a
file name, and click Save.
3. To open image data:


Click the Open button
.
In the dialog that appears, select the data of interest and
click Open.
Figure 5-7 Plate results (plate.txt) provide a summary of the data (per
channel) for the entire plate
96
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Table 5-2 Plate results (plate.txt)
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Item
Description
Number of Objects
Total number of objects in the well that were identified by
the process.
Area
Sum total of the area of all objects in the well.
Area Pix
The total area of all objects in the region of interest (or
the entire well image if there is no region of interest)
which is quantified by the number of pixels in the object.
You will need to use the area of the pixel to convert to
the physical area parameter.
Shape Factor
Average object perimeter.
Mean Intensity
Average object intensity.
Weighted Mean
Intensity
The mean intensity of each object in the ROI multiplied
by its area, divided by the area of all the objects in the
ROI. This enables quantitation of a mean intensity that is
weighted by the size of the object.
Peak Intensity
The average of all the peak pixel intensities of every
object in the region of interest that has passed the
particle or object filters.
Total Intensity
Sum total of the intensity of all objects in the region of
interest (or the entire image of the well if there is no
ROI).
Background
Intensity
Average background intensity in the well. Background
pixels are all those that fall below the Threshold level
specified in the Process.
97
Viewing Data Files
Figure 5-8 Well results (.csv) provide information about each particle in
the well
98
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Table 5-3 Well results (.csv)
Item
Description
PID
Particle identification number
X
x-coordinate of the particle [μm]
Y
y-coordinate of the particle [μm]
AREA
Particle area [μm2]
AREA-PIX
The number of pixels in the particle.
SHAPE
FACTOR
Perimeter of the particle.
ANI-1
Calculated anisotropy (channel 1 and 2)
ANI-2
Calculated anisotropy (channel 3 and 4)
IMEAN-1
Average pixel intensity in the particle.
Note: The number (1, 2, 3, or 4) that follows the data
name indicates the channel. The number 5 or 6 indicates
data generated after a formula was applied during
processing.
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IPEAK-1
Peak pixel intensity in the particle.
IINT-1
Total integrated pixel intensity in the particle.
IBGND-1
Background intensity in the entire image.
99
Viewing Data Files
100
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ImageXpress Velos Cytometer
Data Acquisition Quick Start Guide
6
Step 1: Verify Filters
Make sure that the dichroic mirrors and filters match the fluorophores
in the assay protocol.For more details see Verify Emission Filters on
page 27.
Table 6-1 ImageXpress® Velos Cytometer filters
Laser
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Dichroic
Mirror
Filters
405 nm 495 DRLP
430–480 BP, 510–540 BP
440 nm 510 DRLP
460–500 BP, 525 LP
488 nm 560 DRLP
510–540 BP, 560–610 BP, 560–580 BP, 600 LP,
650 LP
532 nm 610 DRLP
560–610 BP, 660–680 BP
640 nm 690 DRLP
660–680 BP, 690–800 BP
101
ImageXpress Velos Cytometer Data Acquisition Quick Start Guide
Step 2: Calibration (optimize PMT & focus settings)
Note: This step is not needed if you are using an existing method with
a routine assay.
1. Turn on the ImageXpress® Velos Cytometer.
It takes 5 minutes for the laser to warm up and 30 minutes for
the instrument to stabilize.
2. Open the ImageXpress Velos Software.
3. Eject the plate nest by clicking the blue up-arrow in the toolbar.
4. Insert the microplate with well A1 in the upper-left corner.
5. Load the plate nest by clicking the blue up-arrow in the toolbar.
6. Open the method that corresponds to the assay protocol.
7. To start calibration, click Calibration > Calibration.
8. In the Go to Column field, type the column number and click Set.
9. Click the blue “start” arrow in the Calibration toolbar to view
data traces.
10. In the Display Channel area, select the channels to be viewed.
11. Make any required Channel Adjustments:
 Channel: Select the PMT channel to adjust.
 PMT Gain: Type new PMT value and click Set.
 Amplitude of signal should be typically 50% of vertical scale.
102
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12. Adjust the Collection Focus up and down in 100 μm increments to
verify that the signal is maximized. For more details on
adjusting the focus, see Step 5: Adjust the collection or
scanning focus on page 48.
13. Click Update Method Home to save the changes to the current
method.
14. Stop the Scan by clicking the blue square in the Calibration
toolbar and then exit Calibration.
Step 3: Acquire and process data simultaneously in
ImageXpress Velos
1. In Instrument Setup, select the laser excitation you will be using
for the assay.
2. Check the resolution setting and detector setup. Confirm that
the correct laser is selected.
3. Verify the Analysis setup.
4. Verify that the Enable Image Analysis check box is selected.
5. Verify that the correct process and post process files are
selected.
6. Verify the data file types to be saved.
7. Select wells. Hold down the left mouse button and drag to select
wells. Hold down the right mouse button and drag to deselect
wells.
8. Start the scan by clicking the blue arrow.
9. Save the data. Data is saved in directories with the user file
name.
 .bbd: raw data file or method
 .csv: individual well object list
 .txt: plate results file
 .fcs: flow cytometry standard file
 .tif: image file
 .iso: FCS Express compatible
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103
ImageXpress Velos Cytometer Data Acquisition Quick Start Guide
104
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ImageXpress Velos Software
Menu Commands and Toolbar
A
Toolbar
Figure A-1 ImageXpress® Velos Software toolbar
The commands available in the toolbar change depending on whether a
plate map window or image window is open and selected.
File Menu
Table A-1 ImageXpress Velos Software File menu commands and
toolbar buttons
Menu Command
Toolbar Description
Button
File > New
Opens the Instrument Setup dialog (see
page 32) that enables you to specify a
method (acquisition parameters, a process,
and the types of results to generate when
you save the image data).
File > Open Data
Opens a dialog that enables you to select
image data (.bbd).
File > Open Method
Opens a dialog that enables you to select
method (.bbd).
File > Save Data As
Opens a dialog that enables you to save
image data (.bbd).
File > Save Method
As
Opens a dialog that enables you to save a
method (.bbd).
Note: Save the image data before you
save the method. Otherwise, the data
will be discarded when you save the
method.
File > Close
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Closes the image data (.bbd).
105
ImageXpress Velos Software Menu Commands and Toolbar
Table A-1 ImageXpress Velos Software File menu commands and
toolbar buttons (cont’d)
Menu Command
Toolbar Description
Button
File > Load Analysis
Results
Opens a dialog that enables you to select a
.bba file. The .bba file includes the image(s),
the associated well data (.csv), and the
process that was used in the analysis.
File > Exit
Closes the ImageXpress Velos Software.
Image Menu
Table A-2 ImageXpress Velos Software Image menu commands and
toolbar buttons
Menu Command
106
Toolbar Description
Button
Image > Channel __
Selects the data to display in the open
image: data acquired using channel 1, 2, 3,
4, 5 (1 & 2), or 6 (3 & 4).
Image > Composite
Shows the sum of the images from two
channels (1 and 2 or 3 and 4).
Image > Brightness
and Contrast
Displays the controls for adjusting the image
appearance.
Image > Region of
Interest (ROI)
Displays the tools that you can use to place
an ROI on the image.
Image > Options
Enables you to select the display color in the
well image for particles identified by the
analysis and particles highlighted in the .csv
file. Enables you to show or hide the image
dimensions and pixel size in the image.
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Tool Menu
Table A-3 ImageXpress Velos Software Tool menu commands and
toolbar buttons
Menu Command
Toolbar Description
Button
Tool > Pan
Selects the
tool in the image window that
enables you to move the image in the image
window.
Tool > Zoom
Selects the
tool in the image window
that enables you to magnify an image.
Tool > Select
Selects the
tool in the image window that
enables you to select a particle.
Tool > Zoom Out
Selects the
tool in the image window
that enables you to reduce the image
magnification.
Tool > View All
Resets the image magnification so that the
entire well contents are displayed.
Analysis Menu
Table A-4 ImageXpress Velos Software Analysis menu commands and
toolbar buttons
Menu Command
Analysis > Process
Toolbar Description
Button
Opens the Analysis Processing control panel.
Note: This button is available only when
a well image is open and selected.
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Analysis > Summary
Opens the Particle Summary information
(.csv).
Analysis > Columns
Opens that Particle Columns dialog that
enables you to select the information to
display in the Particle Summary (.csv)
107
View Menu
Table A-5 ImageXpress Velos Software View menu commands and
toolbar buttons
Menu Command
View > Enlarge
Toolbar Description
Button
Increases the size of the plate map.
Note: This command is available only
when an image data file (.bbd) is open.
View > Reduce
Reduces the size of the plate map.
when an image data file (.bbd) is open.
View > Instrument
Setup
Opens the Instrument Setup dialog.
View > Toolbar
Shows or hides the ImageXpress Velos
Software toolbar.
View > Status Bar
Shows or hides the status bar at the bottom
of the main window.
when a method or image data file
(.bbd) is open.
Scan Menu
Table A-6 ImageXpress Velos Software Scan menu commands and
toolbar buttons
Menu Command
108
Toolbar Description
Button
Scan > Eject Plate
Moves the scanner plate holder to the
outside of the instrument so that you can
load a plate.
Scan > Load Plate
Retracts the scanner plate holder into the
instrument.
Scan > Start Scan
Starts a scan using the open method.
Scan > Abort Scan
Stops the scan that is currently in progress.
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Data Menu
Table A-7 ImageXpress Velos Software Data menu commands and
toolbar buttons
Menu Command
Toolbar Description
Button
Data > Analyze Data
Analyzes or reanalyzes the scan data using
the process specified in the method.
Data > Export Tiff
Images
Opens a dialog that enables you to batch
export the selected wells from the .bbd data
file to individual tiff files.
Data > Discard Data
Permanently removes all data associated
with the method.
Data > Close All
Images
Closes all well images.
Window Menu
Table A-8 ImageXpress Velos Software Window menu commands and
toolbar buttons
Menu Command
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Toolbar Description
Button
Window > Cascade
Arranges well images and the plate map in a
cascade in the main window.
Window > Tile
Horizontal
horizontal tile in the main window (top to
bottom).
Window > Tile
Vertical
vertical tile in the main window (left to
right).
109
Autorun Menu
Table A-9 ImageXpress Velos Software Autorun menu commands and
toolbar buttons
Menu Command
Toolbar Description
Button
Autorun >
Setup/Run
Opens a dialog that enables you to setup and
start an autorun.
Autorun > Advanced
Opens the Autorun Advanced Settings dialog.
Autorun > Batch
Process
Opens the Batch Process dialog. This enables
you to automatically re-analyze a number of
data files with a new Process and save the
data. This is helpful when you want to run
large numbers of plates (screening
environments) and you want to change the
image process parameters.
Control Menu
Table A-10 ImageXpress Velos Software Control menu commands and
toolbar buttons
Menu Command
Toolbar Description
Button
Control > Local
Allows the user to operate the software on
the current computer.
Control > Remote
Allows the user to operate the software on
another computer remotely.
Help Menu
Table A-11 ImageXpress Velos Software Help menu commands and
toolbar buttons
Menu Command
Help > About
ImageXpress Velos
110
Toolbar Description
Button
Displays the software version information.
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B
ImageXpress Velos Cytometer Files
Table B-1 ImageXpress® Velos Cytometer files
File Name
Description
See
Page
Image method (.bbd)
Includes the acquisition and, optionally, the analysis process.
31
Note: After a scan, the method includes image data.
“Method derived from”
_____(.bbd)
The method that was used to acquire particular data. It
88
includes all acquisition and analysis settings in the Instrument
Setup dialog. The method is saved independently from the
data. Save the method immediately after you save the data.
Plate analysis results
(.plate.txt)
Contains the analysis results for the entire plate. Summarizes 97
and averages the object measurements within each well. The
plate.txt file can be imported into Excel or other database of
your choice.
Well analysis results (.csv) The analysis results include data for the particles in a single
well (one .csv per well).
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99
111
Table B-1 ImageXpress® Velos Cytometer files (cont’d)
File Name
Description
See
Page
Process (.bbp)
Specifies the parameters that the ImageXpress Velos
Software uses to analyze an image file.
55
Analysis results (.bba)
Includes one or more user-selected well images and
associated analysis results (.csv).
81
Post-process (.ppr)
A post-process for validated applications.
37
112
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Table B-1 ImageXpress® Velos Cytometer files (cont’d)
File Name
Description
FCS compatible file (.fcs)
The Flow Cytometry Standard file format. Files in this format
can be viewed using third-party software (for example,
FlowJo, FCS Express). One .fcs file is generated per well if you
choose the option “Produce individual fcs files” in the
Instrument Setup dialog.
FCS Express compatible
file (.iso)
The Flow Cytometry Express file format. Files in this format
can be viewed using FCS Express. One .iso file is generated
per well if you choose the option “Produce individual iso files”
in the Instrument Setup dialog.
MDCStore™ compatible
file
A file that is compatible with the MDCStore database (one
image file (.tif) per well per wavelength and one header file
(.htd) per plate). Files in this format can be imported into the
MDCStore database and analyzed using MetaXpress®
Software.
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See
Page
113
114
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C
Validated Fluorophores and
ImageXpress Velos Filters
Table C-1 Validated fluorophores for the 488 nm laser
Spectrum
Green
Orange
Red
Far Red
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Fluorescent Label
Wavelength (nm)
Excitation
Emission
ImageXpress® Vel
os Filter
Acridine Orange
500
526
510–540
Alexa Fluor 488 (AF–488)
495
519
510–540
BODIPY–FL
505
513
510–540
Calcein AM
494
517
510–540
Chromeo–488
488
517
510–540
CMFDA
492
517
510–540
Cy2
489
506
510–540
EGFP
488
509
510–540
EYFP
513
527
510–540
Fluorescein (FITC)
494
517
510–540
Fluo–4
494
516
510–540
5–FAM
(5–Carboxyfluorescein)
492
518
510–540
JC–1
498
525
510–540
Rhodamine 110
496
520
510–540
Rhodamine 123
507
529
510–540
SYTOX Green
504
523
510–540
YO–PRO–1
491
509
510–540
Alexa Fluor 532
532
553
560–610
PE (R–Phycoerythrin)
565
575
560–580
Ethidium homodimer–1
(EthD–1)
528
617
600 LP
LavaCell (Epicocconone)
488
610
600 LP
Propidium Iodide (PI)
536
617
600 LP
7–AAD
(7–Aminoactinomycin D)
546
647
650 LP
115
Validated Fluorophores and ImageXpress Velos Filters
Table C-1 Validated fluorophores for the 488 nm laser (cont’d)
Spectrum
Fluorescent Label
Wavelength (nm)
PE–Cy5
488
670
650 LP
PE–AF647
488
668
650 LP
PerCP
488
675
650 LP
Spectrum
Blue
Green
Fluorescent Label
Wavelength (nm)
Excitation
Emission
ImageXpress® Vel
os Filter
DAPI
358
461
430–480
Hoechst (33342)
352
461
430–480
GeneBLAzer (CCF2)
409
447
430–480
GeneBLAzer acceptor
447
520
430–480
Spectrum
Orange
Red
116
Fluorescent Label
Wavelength (nm)
Excitation
Emission
ImageXpress® Vel
os Filter
Alexa Fluor 532
532
553
560–610
Alexa Fluor 546
556
573
560–610
PE (R–Phycoerythrin)
565
575
560–580
Alexa Fluor 568
579
604
600 LP
Ethidium homodimer–1
(EthD–1)
528
617
600 LP
Propidium Iodide (PI)
536
617
600 LP
Lava Cell (Epicocconone)
535
610
600 LP
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Spectrum
Red
Far Red
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Fluorescent Label
Wavelength (nm)
Excitation
Emission
ImageXpress® Vel
os Filter
Alexa Fluor 647
653
669
680–800
Cy5
649
670
660–680
7–AAD
(7–Aminoactinomycin D)
546
647
680–800
DRAQ5
646
670
650 LP
Alexa Fluor 660
663
690
680–800
Cy5.5
675
694
680–800
117
Validated Fluorophores and ImageXpress Velos Filters
118
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D
Resolution and Typical Scan Times
The acquisition time increases proportionally with averaging.
Table D-1 Resolution and scan time
Microplate Format
(Number of wells)
Resolution
(μm)
96
384
Acquisition
Time (min)
Full Plate
File Size
(MB)
1536
No. of Channels
10 x 10
1–4
1–4
1–4
2
100–460
5x5
1–3a
1–4
1–4
4
400–1700
2.5 x 2.5
1a
1–3
1–4
12*
1600+
*Large
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files might require increased acquisition time.
119
Resolution and Typical Scan Times
120
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Working in Autorun Mode
E
• Setting Up an Autorun with the Twister II Microplate Handler on
page 122
• Setting Up an Autorun to Do Multiple Scans on page 128
• Starting an Autorun on page 134
•
on page 135
In autorun mode, the scanner operates with the Twister® II Plate
Handler (a robotic plate mover) or a simulated robotic plate handler.
Operating the scanner in the autorun mode with a simulated robot
enables you to acquire multiple scans of the same plate without user
intervention and apply up to four different methods to a plate. This
mode is useful for kinetics studies that require multiple plate scans over
a particular time period.
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121
Setting Up an Autorun with the Twister II
Microplate Handler
An autorun configuration file (.cnfg) specifies the autorun settings for
operating the scanner with the Twister II Plate Handler. The following
steps explain how to create and save an autorun configuration file.
1. Click Autorun > Setup/Run on the menu bar.
2. In the Autorun Setup/Run dialog, choose the settings you want
to use. For details on the items in the Autorun Setup/Run dialog,
see Table E-1 on page 123 and Table E-2 on page 124.
Figure E-1 Autorun Setup/Run dialog
122
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Table E-1 Autorun Setup/Run dialog
Item
Description
No. of Cycles
A plate can be scanned up to 41 times. If the cycle number is
greater than one, you must specify the time delay between
scans. Individual delays between scans can be entered
between 1 minute and 4,096 minutes (68 hours).
A warning message appears if the time delay is not long
enough to allow a plate to be scanned and returned to the
destination rack before the next cycle is due.
You can proceed anyway or change the time delay. If you
proceed without increasing the time delay, the plates are
scanned as quickly as the robot can move them into and out
of the ImageXpress Velos Cytometer. The software alerts you
of the difference between your specified time delay and the
actual delay between cycles after each plate scan.
Abort on Error If this option is selected, the autorun program stops if a fatal
error occurs during the run. The error is recorded to an
Autorun log file and all plate activity stops until you restart the
program. If this option is not chosen, the run continues if an
error occurs and the error is recorded in a log file.
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Use Barcodes
If this option is chosen and a barcode reader is detected on
the system, each plate is scanned by the barcode reader
before being placed into the ImageXpress Velos Cytometer
plate tray. For the barcode readers and formats that are
supported by this software, please contact Molecular Devices
support.
Save Data to
Folder
Specifies a folder in a directory where the image and data files
are saved when the scan and analysis are completed. The
method that is used to acquire the data specifies the data files
that are saved (see Step 3: Set the analysis parameters on
page 35). The name of the file will be the Method name plus a
time stamp of 23 characters.
Nest
The ImageXpress Velos instrument (plate nest).
Barcode
The barcode reader station (if present) for scanning a
microplate.
Lid
The de-lidding area.
Setup
Click to open the Setup Rack options dialog for the selected
rack. See Table E-2 on page 124.
Autorun
Configuration
File
Open: Click to open a dialog that enables you to select an
autorun configuration file (.cfg). The file specifies the rack
setup information.
Save: Click to open a dialog that enables you to save the rack
information to an autorun configuration file (.cfg).
123
Table E-2 Setup Rack Options dialog
Item
Description
No. of Plates
The number of plates to be automatically scanned and
delivered to the destination rack.
Do All
Choose this option to scan and deliver all plates in the rack to
the destination rack
Restack
Choose this option to return the plates to the source rack in
the original order after scanning on the ImageXpress Velos
Cytometer.
Note: Restack is automatically enabled if the number of
cycles is more than one. One of the racks must be left
empty or at least partially empty so that it can receive
finished plates before restacking.
Remove Lids
This option is available if a de-lidding station is detected. If
this option is chosen, the plate lid is removed before loading
into the ImageXpress Velos Cytometer. Since the
ImageXpress Velos instrument reads the bottom of the plate,
it is generally unnecessary to remove the plate lid before
scanning.
Method file(s)
Click Browse to select up to four methods for reading,
analyzing, and saving the data from each plate.
Note: Since engaging the polarization filters is a
manual operation, all methods must use the same
polarizer filters.
Note: The method directory path and method file name
cannot include more than 32 alphanumeric characters.
Spaces and file extensions are included in the character
count.
Save Changes Click to save the settings in the Setup Rack Options dialog for
the selected rack. Click Close if you chose a previously saved
Autorun configuration file and did not modify the rack setup.
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3. Click Setup for a rack that you plan to use.
The Setup Rack Options dialog appears.
Figure E-2 Setup Rack options
4. Click Save Changes.
Note: Due to a limitation in Microsoft Windows, store Method
Files, Autorun configuration files, and Data output files and
folders in the C drive main directory. Keep the folder and file
names short. If the file pathway exceeds a certain number of
characters, ImageXpress Velos Software displays an error
message.
5. Repeat step 3 through step 4 for each rack that you plan to use.
Note: At least one rack must remain empty (without setup
information) to provide a destination rack for plates after
scanning is completed.
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125
6. Click Browse and select a destination folder for the scan data.
Note: For each plate scanned, a folder is created that contains all
of the file types specified in the Method used for the Autorun.
The folders are automatically saved and named as
methodname.barcode.datetime.
Figure E-3 Saving the scan data
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7. After you enter the setup information for the racks that you plan
to use, save the autorun configuration.
Figure E-4 Saving the autorun configuration
8. Click Save.
9. Select a directory for the autorun configuration, enter a name
for the autorun configuration file, and click Save.
For details on starting the autorun, see Starting an Autorun on
page 134.
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127
Setting Up an Autorun to Do Multiple Scans
2. Enter the number of cycles (scans).
Figure E-5 Autorun Setup/Run dialog
3. If you are running multiple cycles, click Time Delay in the
Autorun Setup/Run dialog to set the time interval between
scans.
The Time Delay(s) dialog appears. See Figure E-6 on page 129.
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0270-00007 D
Figure E-6 Time Delay(s) dialog
4. In the Time Delay(s) dialog, enter the minutes for each cycle,
and then click Save Changes.
5. Click Setup for Rack 1.
The Setup Rack Options dialog appears. See Figure E-7 on
page 130.
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129
Figure E-7 Setup Rack Options
6. Type the number of plates to be read. To scan a single plate
multiple times, enter 1. To scan an entire rack of plates multiple
times, enter all the plates in the rack.
7. To select the scanning method:
 Click Browse.
 Select a method for scanning and click Open.
To select another method, click Browse again.
Note: To collect kinetic data as quickly as possible, apply the
same method to just 1 plate (up to 41 scans per plate). This
eliminates the time required to move the plate with the robot.
8. Click Save Changes in the Setup Rack Options dialog.
9. In the Autorun Setup/Run dialog, click Browse and select a
destination folder for the scan data.
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.
Figure E-8 Select a folder for the scan data and the configuration
file
10. Click Save and in the dialog that appears, select a destination
folder and enter a name for the configuration file.
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131
Setting Up Autorun with a Simulated Robot
Using a simulated robot is useful when you want to scan a single plate
multiple times.
1. Click Autorun > Advanced on the menu bar.
The Autorun Advanced Settings dialog appears.
Figure E-9 Autorun Advanced Settings
2. Enable the components you want to simulate: Check Robot and
Plate Eject/Retract. For more information, see Table E-3 on
page 132.
Table E-3 Autorun advanced settings
132
Item
Description
Barcode
Reader
Specifies the serial port for the barcode reader. Please contact
technical support for details on the appropriate
communication port setting for your barcode reader.
No. of Racks
The number of racks that are configured for use in the
Twister® II Plate Handler. Up to nine racks can be configured.
Robot
Choose this option to simulate a robotic plate handler.
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Table E-3 Autorun advanced settings (cont’d)
Item
Description
Plate
Eject/Retract
If this option is chosen, the plate remains inside the
ImageXpress Velos Cytometer for all methods and cycles.
Scan Tool
ImageXpress Velos Cytometer. This setting simulates the
instrument, and it is not recommended for normal operations.
For more information, contact technical support.
Save Changes Click to save any changes to the autorun advanced settings.
Close
Click to close the Autorun Advanced Settings box.
3. Click Save Changes.
4. Set up the Autorun as described in Setting Up an Autorun to Do
Multiple Scans on page 128. Manually load a plate into the plate
tray.
Note: During a simulated autorun, only Rack 1 is used.
5. Start Autorun. See Starting an Autorun on page 134.
The ImageXpress Velos Software begins operation in robotic
simulation mode.
Note: The software assumes that racks without setup information do
not contain plates. The first rack without setup information is
designated the output or destination rack for the plates. Racks without
setup information are marked with a dark green indicator ; racks
with setup information are marked with a bright green indicator . If
you chose the Restack option or entered >1 for the number of cycles,
the destination rack is marked by a yellow indicator during the run. If
all of the racks have setup information, an error message informs you
that a destination rack must be left empty.
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133
Starting an Autorun
2. On the Autorun Setup/Run dialog, click Run
. See
Figure E-10 on page 135.
3. A plate map appears and shows the scan progress.
Note: For an autorun with a simulated robotic plate handler,
manually place the plate in the scanner, then start the autorun.
Pausing an Autorun
2. On the Autorun Setup/Run dialog, click Pause
.See
The plate handler parks in the “safe” position after it completes
the movement or scan that is in progress.
Note: When a plate is being scanned or when Autorun is paused,
you can open a well image and view the data for the current
scan. This is a helpful way to confirm that the scanning and data
collection are proceeding as expected. If you cannot see the well
image, you might need to move the Autorun Setup/Run window
out of the way.
3. To restart the autorun, click Run
.
Aborting an Autorun
2. On the Autorun Setup/Run dialog, click Abort
. See
The autorun stops. The robot arm completes the current step
and stops in a safe position.The Autorun/Setup Run window
closes.The status of the ImageXpress Velos Cytometer, plate
handler, and the destination directory of the run log file are
displayed.
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Note: An aborted autorun cannot be resumed. If an autorun is
aborted when a plate is being scanned, the data from the plate
will not be saved. The data from plates that completed scanning
before the Abort command is saved.
Figure E-10 Run, Save, and Abort controls on the Autorun Setup/Run
dialog
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135
136
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ImageXpress Velos Image Registration
Alignment Procedure
F
Topics
•
•
•
•
•
in this section:
Preparing for the New Alignment Plate on page 137
Preparing for Calibration on page 137
Well Alignment Calibration of Laser 1 (488 nm) on page 140
Image Adjustment with the Alignment Plate on page 146
Well Alignment Calibration of Laser 2 (405, 440, 532, or 640 nm
wavelength) on page 150
This appendix explains the procedure for performing registration of
multiwell plates to the center of the ImageXpress® Velos Software
image window (LUT Calibration) and checking alignment of the
ImageXpress Velos Cytometer with the alignment plate.
For information on performing registration with a bead plate, see
ImageXpress Velos Image Registration Alignment Procedure with Bead
Plate (Legacy Plastic Plate) on page 153.
This is an instrument operation and software procedure for performing
a LUT calibration so that wells are properly registered in their image
window. LUT refers to updates automatically made to the Look Up Table
upon accepting a calibration. This procedure is used in initial setup,
during routine calibration, and in field service.
Preparing for the New Alignment Plate
The verification test requires the ImageXpress Velos Software version
5.1 or later, which requires additional calibration information.
The required information is installed with this version of the software at
the time of the software installation. If you are using the new alignment
plate, you must have this version of the software installed or the
calibration will not work according to the calibration procedure
described in this appendix.
Preparing for Calibration
This procedure is for calibration with the 488 nm line. This calibration
procedure can be extended to any of the other laser lines by changing
to the appropriate bandpass filter and dichroic in step 4.
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ImageXpress Velos Image Registration Alignment Procedure
To prepare for calibration:
1. Locate the alignment plate supplied with the ImageXpress Velos
Cytometer.
2. In Table F-1, enter the plate’s serial number and the values
provided on the specification certificate: Edge to First Rule, Rule
Spacing, and Left Offset in units of microns.
Table F-1 ImageXpress Velos Cytometer alignment information
Item
Description
v.5.1 or later
Alignment Plate Serial Number
Edge to First Rule Value from the alignment
plate certificate (μm)
Rule Spacing Value from the alignment plate
certificate (μm)
Left Offset Value from the alignment plate
certificate (μm)
There is a ruling in the center of the bottom of the plate.
3. Inspect the plate to see that the ruling is not damaged or that it
does not contain any extraneous marks.
CAUTION! Do not touch the ruling and the bottom of the plate with your
fingers to keep the plate clean from fingerprints and dust. To clean the
bottom of the plate, use only lens tissue moistened with isopropanol.
4. Insert 510–540 nm filters in Channel 1 and 2. Use standard
560DRLP dichroic mirrors.
5. Close the light-tight filter door and the external filter door.
6. Start the ImageXpress Velos Software from the desktop
shortcut.
7. From the toolbar, click the upward-pointing arrow to open the
plate nest.
8. Place the alignment plate in the plate nest with the plate’s ruling
facing down and the plate’s A1 position in the top-left corner.
9. Open Method for Alignment Plate, if it is already present. If the
Method for Alignment Plate is not present, see Creating a
Method for Alignment Plate on page 139.
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Creating a Method for Alignment Plate
If the Method for Alignment Plate is not present, create a method with
the following settings:
• Plate Type: ImageXpress Velos Alignment Plate file that matches
the plate you are using. See the specification certificate for the
plate.
• Scan Area: 3200 x 3200 μm
• Scan resolution: 10x10 μm
• Data Averaging: 1 avgs/pixel
• Channel 1: Enabled, Green All, Gain between 200–700 mV,
Sensitivity 1X (nominally 500)
Sensitivity 1X (nominally 500)
• Channel 3: Not Enabled
• Channel 4: Not Enabled
• Method's Collection Focus: –100 μm to +100 μm
• Method's Scanning Focus: –100 μm to +100 μm
• Image Analysis: Not Enabled
• Produce data (.bbd) file: Enabled
• Auto. Correct Saturated Data: Enabled
• Leave all other choices for analysis unselected
Use this method to acquire alignment data. You can optimize the PMT
gain and the focus settings for the particular plate that you are using.
The gain is nominally in the 500 mV range. If it needs to be set above
700 mV, there might be a problem with the alignment plate.
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139
Well Alignment Calibration of Laser 1 (488 nm)
1. Prepare for the calibration as described in Preparing for
Calibration on page 137.
2. Open or create the Method for Alignment Plate as described in
Creating a Method for Alignment Plate on page 139.
3. From the menu bar, click Calibration > Calibration to open the
Calibration window.
4. In the Scope Window area, verify that Display Channel 1 and 2
are selected.
5. In the Go to column field, type either 11, 12, 13, or 14 and click
Set.
Note: 11, 12, 13, and 14 represent column numbers on a 384well plate and correspond with the location of the ruling on the
bottom of the alignment plate. Over time and use, areas of the
of the alignment plate can become photobleached. Type the
column number that gives you the best calibration result.
6. Verify that the PMT gain setting for Channel 1 and Channel 2 are
between 200 mV and 700 mV.
7. Leave the values that are in the Edge to First Rule, Rule Spacing,
and Left Offset fields as they are unless you are working with a
new alignment plate.
If you are working with a new alignment plate, type the values
from the specification certificate in the Edge to First Rule, Rule
Spacing, and Left Offset fields.
8. Type the Cal Password: isct.
This password is case-sensitive.
9. Click the Scan button in the top-left corner of the window.
The text of the Calibrate button turns from gray to black if the
password is entered properly. If the text on Calibrate button
remains gray, then stop the scan and retype the password.
The ruling signals appear as a series of peaks on the calibration
screen. The peak locations are used to calibrate the well
registration.
10. Change the value in the PMT gain field for Channel 1 and
Channel 2 so that most of the peaks reach between 50% and
75% of the full scale on the screen but do not go above 100% of
the scale. See Figure F-1.
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Figure F-1 Example calibration screen
11. While the system is scanning, click Calibrate.
The Calibration Time Delay dialog appears. Calibration begins
when the countdown clock reaches 0.
Figure F-2 Calibration Time Delay dialog
When the calibration is complete, the Calibration Result dialog
appears.
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141
Figure F-3 Calibration Result dialog
12. To view details of the calibration, click Show Details.
Figure F-4 Calibration Result dialog with details
13. If the calibration is successful, click Accept.
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Note: In general, the max/min ratio can be high without causing
calibration issues. However, if later you have trouble centering
the well image, you might need to contact technical support.
14. If the calibration fails, click Show Details to view details.
15. To resolve the failure, click Cancel and follow these
recommendations:
 Make sure a clean alignment plate is being used and that the
values for Edge to First Rule, Rule Spacing, and Left Offset in
the Calibration window match the values on the specification
certificate for the plate.
 Make sure the peak signals during the scan are between
50% and 75% and that the Collection and Scanning Focus
are optimal.
After taking the corrective actions, repeat step 8 through
step 13.
Figure F-5 Calibration Result dialog showing failure
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143
Figure F-6 Calibration result dialog with details of failure
16. After accepting the new calibration, click Apply n Quit at the
bottom of the Calibration dialog.
The Calibration dialog closes and the new calibration values are
registered.
17. In the menu bar, click File > Save Method As and save the method
using the same file name.
18. In the Plate Map window, select column 1 and then click the
Scan button in the top-left corner of the window.
19. Double-click well A1 to open the A1 Image window.
20. Verify that the image is centered in the window (Figure F-7).
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Figure F-7 Example of A1 Image centered in window
21. If the image is acceptable, then double-click well P1 to open the
P1 Image window and verify that the image is centered in the
window.
If either image is not centered in the window, see Image
Adjustment with the Alignment Plate on page 146.
22. If both images are centered, close the well image windows.
Calibration window.
If the Calibration menu does not appear in the menu bar, click
the Plate Map window to change the available menu selections.
24. In the message that appears asking if you want to discard your
data, click Yes.
25. Record the values from the Edge to First Rule, Rule Spacing, and
Left Offset fields in Calibration log (Table F-3 on page 151).
The Calibration is finished.
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145
Image Adjustment with the Alignment Plate
When performing the LUT calibration, you can register the wells using
the columns 1 or 2 and 23 or 24 of the alignment plate, if the
appropriate alignment plate file has been supplied with the alignment
plate.
Use the following procedures to register the wells:
• If the image of A1 is too high in the window (shifted up toward
the top): on page 146
• If the image of A1 is too low in the window (shifted toward the
bottom): on page 146
• If the image of A1 is too far left in the window: on page 147
• If the image of A1 is too far right in the window: on page 147
• If the image in A1 is centered, but images down the column are
incrementally shifting: on page 148
You can also register the wells using a bead plate. For the image
adjustment procedure using a bead plate, see Image Adjustment with
Bead Plate on page 159.
If the image of A1 is too high in the window (shifted up
toward the top):
1. Decrease the value in the Edge to the First Rule field by 50 μm to
500 μm (depending on severity of image shift).
2. Repeat the calibration procedure until A1 is centered. See Well
Alignment Calibration of Laser 1 (488 nm) on page 140.
If the image of A1 is too low in the window (shifted
toward the bottom):
1. Increase the value in the Edge to the First Rule field by 50 μm to
500 μm (depending on severity of image shift).
2. Repeat the calibration procedure until A1 is centered as shown
in Figure F-8. See Well Alignment Calibration of Laser 1
(488 nm) on page 140.
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Before
After
Figure F-8 Image that registered too low was corrected by
decreasing the Edge to first Rule Value by 500 μm
If the image of A1 is too far left in the window:
1. Decrease the value in the Left Offset field by 50 μm to 500 μm
(depending on severity of image shift).
If the image of A1 is too far right in the window:
1. Increase the value in the Left Offset field by 50 μm to 500 μm
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147
If the image in A1 is centered, but images down the
column are incrementally shifting:
1. Close the well image windows.
4. Double-click well P1 to open the P1 Image window.
5. From the P1 Image window toolbar, click the ROI button.
6. In the Region of Interest dialog, select the Rectangular option.
7. In the X center offset and Y center offset fields, type 0 (zero).
8. In the Width and Height fields, type 750.
9. Drag and resize the ROI rectangle around the center box in the
P1 Image window as in Figure F-9.
 If the Y center offset value is greater than 50 μm in P1,
increase the Rule Spacing value by 0.5 μm to 2μm in the
Calibration window (follow step 10 through step 12).
 If the Y center offset value is less than –50 μm in P1,
decrease the Rule Spacing value by 0.5 μm to 2μm in the
Calibration window (follow step 10 through step 12).
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ROI Rectangle
Figure F-9 ROI box defining the center box in P1 Image window.
Calibration window.
11. Type the new value in Rule Spacing field.
Changing the offset value by 1 μm shifts the image in P1
approximately 100 μm.
12. Run a calibration scan with the new value. See Well Alignment
Calibration of Laser 1 (488 nm) on page 140.
13. Repeat step 1 through step 9 until the Y center offset value in P1 is
between –50 μm and 50 μm.
14. Write down the final calibration values for the 488 nm laser in
the Calibration log (Table F-3 on page 151).This record will be
useful to Molecular Devices service engineers in the future.
To complete the ILUT calibration, see ImageXpress Velos Intensity
Calibration Procedure on page 165.
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149
(405, 440, 532, or 640 nm wavelength)
1. Make sure that the filters in Channel 1 and 2 and dichroic
mirrors match the wavelength of the Laser 2 (see Table F-2).
Table F-2 Matching laser and dichroic mirror
2.
3.
4.
5.
6.
7.
8.
Laser (nm) Ch1 (nm) Ch2 (nm)
Dichroic Mirror (nm)
405
430–480
430–480
495
440
460–500
460–500
510
532
560–610
560–610
610
640
690-800*
690-800*
690
*For general acquisition, the filter setting for the 640 nm laser is
660-800 for channels 1 and 2. For calibration, however, the
setting is 690-800 for channels 1 and 2.
Open Method for Alignment Plate.
From the menu bar, click View > Instrument Setup.
In the Instrument Setup dialog, click the Detect tab.
Select the Laser 2 option.
Click OK.
From the menu bar, click Calibration > Calibration to open the
Calibration window.
The Laser 2 wavelength appears on the Calibrate button.
Follow the Calibration procedure used for Laser 1. See Well
Note: The PMT gains for Laser 2 can be higher than those used for
Laser 1 to achieve a 50% signal in the scope window.
To complete the ILUT calibration, see ImageXpress Velos Intensity
Calibration Procedure on page 165.
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Table F-3 Calibration log
Calibration
Date
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Edge to
First Rule
(μm)
Rule
Spacing
(μm)
Left
Offset
(μm)
CH 1
PMT Gain
(mV)
CH 2
PMT Gain
(mV)
Operator
Initials
151
152
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ImageXpress Velos Image Registration
Alignment Procedure with Bead Plate
(Legacy Plastic Plate)
G
Topics
•
•
•
•
in this section:
Preparing for Calibration on page 153
Well Alignment Calibration of Laser 1 (488 nm) on page 155
Image Adjustment with Bead Plate on page 159
Well Alignment Calibration of Laser 2 (405, 440, 532, or 640 nm
wavelength) on page 161
This appendix explains the procedure for performing registration of
multiwell plates to the center of the ImageXpress® Velos Software
image window (LUT Calibration) and checking alignment of the
ImageXpress Velos Cytometer using a Bead Plate.
For information on performing registration with the alignment plate,
see ImageXpress Velos Image Registration Alignment Procedure on
page 137.
a LUT calibration so that wells are properly registered in their image
window. LUT refers to updates automatically made to the Look Up Table
upon accepting a calibration. This procedure is used in initial setup,
during routine calibration, and in field service.
Cytometer.
2. In Table G-1, enter the plate’s serial number and the values
provided on the label: Edge to First Rule and Rule Spacing in units
of microns.).
Table G-1 ImageXpress Velos Cytometer alignment information
Item
Description
v.5.0 or later
Alignment Plate Serial Number
Edge to First Rule Value from Plate Label (μm)
Rule Spacing Value from Plate Label (μm)
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ImageXpress Velos Image Registration Alignment Procedure with Bead Plate (Legacy Plastic Plate)
3. You will notice a ruling on the bottom of the plate. Inspect to see
that the ruling is not damaged or contains any extraneous
marks.
Note: The ruling and the bottom of the plate in general should
not be touched by fingers in order to keep the plate clean from
fingerprints and dust. Do not clean bottom of plate with liquid as
this will damage the alignment plate.
4. Make sure that the filters in Channel 1 and 2 are 510–540 nm
bandpass and the dichroic mirror is 560 nm for calibrating Laser
1 (normally 488 nm).
5. Open the ImageXpress Velos Software.
6. Open Method for Alignment Plate. Example ranges for a Method
for Calibrating the LUT for a 488 nm laser is shown below.
Plate Type: 384 Matrical
Scan Area: 3200 x 3200 μm
Scan resolution: 10x10 μm
Data Averaging: 1
Channel 1: Enabled, Gain between 300–600 mV, Polarization All
Channel 2: Enabled, Gain between 300–600 mV, Polarization All
Laser 1 (488 nm): Enabled
Method's Collection Focus: –600 to –900 μm
Method's Scanning Focus: –600 to –900 μm
Analysis: Nothing is enabled
7. Load the alignment plate in the ImageXpress Velos Cytometer
with well A1 in the standard orientation.
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Well Alignment Calibration of Laser 1 (488 nm)
1. Load the alignment plate and open the Method for Alignment Plate
as described in Preparing for Calibration on page 153.
Calibration window.
3. In Scope Window, verify that Display Channel 1 and 2 are
checked.
4. Go to column 11, 12, 13, or 14 (where rulings are located on
Alignment Plate) and click Set.
5. Verify that the PMT gain setting for Ch 1 and Ch 2 are between
300–600 mV.
6. If this is the initial instrument calibration, enter the Edge to First
Rule and Rule Spacing numbers provided in the locations below
the Calibrate button.
7. If this is a re-calibration using the same plate, leave the
numbers that are currently in the Edge to First Rule and Rule
Spacing boxes.
8. Enter the Cal Password: isct.
You will see the ruling signals as a series of peaks on the
calibration screen. The peak locations will be used to calibrate
the well registration.
10. Select the Use Legacy Plastic Plate box.
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155
11. Adjust Channel 1 and Channel 2 gains so that peaks reach
between 50% and 75% of full scale on the screen but do not go
off scale (Figure G-1).
Figure G-1 Example calibration screen
Figure G-2 Calibration Time Delay dialog
13. When the calibration is complete, the Calibration Result dialog
appears.
A window opens and shows the results of the Calibration. Accept
the Calibration if the “number of edges” results in PASS and the
max/min ratio results in PASS. Cancel if “Number of edges”
results in FAIL and follow these recommendations:
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0270-00007 D
Make sure a clean alignment plate with Edge to First Rule
and Rule Spacing values matching those entered in the
Calibration screen is being used.
 Make sure the peak signals are between 50% and 75% and
the Collection and Scanning Focus are optimal.
 After taking the corrective actions, click Calibrate again.
 If number of edges results in PASS but Max/min ratio is high,
accept the Calibration and continue.
14. After accepting the new calibration, click Update Method Home to
save the changes to the current method.
15. Click Apply n Quit at the bottom of the calibration screen.
16. The Calibration window closes. The new Calibration values are
now entered into the registry.
17. Save the Method using the Save As command to overwrite an
existing Method.
18. Scan column 1 and view well A1 to verify that the image is
centered in the window (Figure G-3).

Note: This Calibration only adjusts the alignment from top to
bottom in the well, not from left to right
Figure G-3 Example of a centered image where the beads are
outlining the sidewalls of the well, visualizing the well in the image
window.
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157
If the image is acceptable, open well P1 and inspect the
alignment.
 If the image is too high or too low, return to the calibration
screen.
 If both images are centered, return to the calibration screen
(discard scan data) and record the numbers from Edge to
First Rule and Rule Spacing areas in Table G-3 on page 164.
The Calibration is finished.

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Image Adjustment with Bead Plate
To adjust the Y location of the image in well A1, change the value in
Edge to First Rule.
If the image is too high in the window (shifted up toward
the top):
1. Increase the Edge to the First Rule value by 50 μm to 500 μm
2. Repeat System Calibration (step 8 to step w) until A1 is
centered.
If the image is too low in the window (shifted toward the
bottom):
1. Decrease the Edge to the First Rule value by 50 μm to 500 μm
2. Repeat System Calibration (step 8 to step w) until A1 is
centered as shown in Figure G-4.
Before
After
Figure G-4 Image that registered too low was corrected by
decreasing the Edge to first Rule Value by 500 μm
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159
If the image in A1 is centered, but images down the
column are incrementally shifting:
1. Adjust the spacing between wells down a column by changing
the value in the Rule Spacing.
2. If the images are shifting lower in the window (Figure 4),
decrease the Rule spacing value by 1 μm or 2 μm. Repeat the
system calibration (step 8 to step w) until wells A1, H1, and P1
are centered.
Figure G-5 Images shifting lower in the window as you scan
down the column (required a 2 μm adjustment to the Rule
spacing)
3. If images are shifting higher in the window, increase the Rule
spacing value by 1 μm or 2 μm. Repeat the system calibration
(step 8 to step w) until wells A1, H1, and P1 are centered.
4. Write down the final calibration values for the 488 nm laser in
the Table G-3 on page 164.This record will be useful to
Molecular Devices service engineers in the future.
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(405, 440, 532, or 640 nm wavelength)
1. Make sure that the filters in Channel 1 and 2 and dichroic
mirrors match the wavelength of the Laser 2 (see Table G-2).
Table G-2 Matching laser & dichroic mirror
Laser
(nm)
Ch1 (nm)
Ch2 (nm)
Dichroic Mirror (nm)
405
430–480
430–480
495
440
460–500
460–500
510
532
560–610
560–610
610
640
660–680
660–680
690
2. Open the Instrument Setup dialog – Detect tab.
 Click Laser 2.
 Click OK.
3. Open the calibration screen (Calibration > Calibration).
Notice that the Laser 2 wavelength is showing in the Calibrate
button.
4. Follow all the same steps for Calibration as used previously for
Laser 1.
Note: The PMT gains for Laser 2 will likely be higher than those used
for Laser 1 to achieve a 50% signal in the scope window (see
Figure G-6).
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161
Figure G-6 Example calibration screen for the 640 nm laser
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Figure G-7 shows an example of aligned well images for Laser 2.
Figure G-7 Example of aligned wells using Laser 2 (640 nm)
The alignment plate contains only red and green beads that may not
show up well if the beads do not excite with the wavelength of Laser 2.
If this is the case, remove the filters from Channel 1 and 2 and use
laser scatter to view the edges of the wells for proper alignment. If the
edges are still difficult to see, scan the plate with focus settings of 0, 0
instead of –700, –700 μm.
• Write down the final calibration values for Laser 2 in Table G-3
on page 164.
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163
Table G-3 Calibration log
Calibration Edge to First Rule Spacing
Date
Rule (μm)
(μm)
164
CH 1 PMT
Gain (mV)
CH 2 PMT
Gain (mV)
Operator
Initials
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ImageXpress Velos Intensity
Calibration Procedure
H
• Preparing for Calibration on page 165
• Intensity Calibration (ILUT) for Laser 1 (typically 488 nm) on
page 166
• Intensity Calibration (ILUT) for Laser 2 on page 175
This appendix describes the procedure for performing intensity
uniformity correction and calibration of the ImageXpress® Velos
Cytometer PMT signals (ILUT Calibration).
an ILUT calibration so that PMT signals are corrected for relative
uniformity across a scan line. This procedure is used in initial setup,
during routine calibration, and in field service. The Image Registration
Alignment Procedure should always be completed before the
ImageXpress Velos Intensity Calibration Procedure. See
ImageXpress Velos Image Registration Alignment Procedure on
page 137.
1. Power on the ImageXpress Velos Cytometer and let it warm up
for at least one hour.
2. Start the ImageXpress Velos Software.
Cytometer.
There is ruling in the center of the bottom of the plate (located
below where columns 11 through 14 are on a 384-well plate)
and solid yellow fluorescent bands on either side (located below
where columns 7 and 8, and 17 and 18 are on a 384-well plate).
4. Inspect the plate to see that the ruling and yellow areas are not
damaged or that they do not contain any extraneous marks.
CAUTION! Do not touch the ruling and the bottom of the plate with your
fingers to keep the plate clean from fingerprints and dust. To clean the
bottom of the plate, use only lens tissue moistened with isopropanol.
5. Insert 510–540 nm filters in Channel 1 and 2 and use a
standard 560DRLP dichroic mirror.
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165
ImageXpress Velos Intensity Calibration Procedure
plate nest.
8. Place the alignment plate in the plate nest with plate’s ruling
facing down and the plate’s A1 position in the top-left corner.
(typically 488 nm)
1. Load the alignment plate in the ImageXpress Velos Cytometer
as described in Preparing for Calibration on page 165.
2. Open or create the Method for Alignment Plate. See Steps to
Create a Method on page 31.
Calibration window.
4. In the Scope Window area, verify that Display Channel 1 and 2 are
selected.
5. In the Go to column field, type either 7 or 8 and click Set.
6. Verify that the Collection Focus and Scanning Focus positions are
between –100 and +100.
7. Verify the PMT gains are between 200 mV and 700 mV.
8. Type the Cal Password: isct
One colored line appears for each channel enabled. You might
need to adjust the PMT gains to get the intensity of both
channels between 25% and 50% of full scale (see Figure H-1).
10. If there are signal spikes recording past the top of the screen,
then in the Motors Jogging area, click the left or right arrow next
to Scan Line to move the Scan Line 100 μm until the spikes
disappear.
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0270-00007 D
Figure H-1 Raw intensity of Channel 1 and Channel 2
Figure H-2 Calibration Time Delay dialog
When the calibration is complete, the Calibration Result dialog
appears.Calibration Result dialog appears.To see the standard
deviation of intensity correction, click Show Details.
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167
Figure H-3 Successful ILUT calibration
The lower the standard deviation, the less correction will be
applied to the raw data. The calibration is acceptable if the
number is less than 0.150000 (15%).
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0270-00007 D
12. Do one of the following:
 If the calibration is acceptable, click Accept.
 If the result is 15% or greater, click Cancel and return to the
Calibration window. In the Motors Jogging area, click the left
or right arrow next to Scan Line to move the Scan Line
100 μm. Repeat step 4 through step 12.
 If you are unable to obtain a result less than 15%, contact
technical support. See Obtaining Support on page 18.
13. Click Apply n Quit at the bottom of the screen to accept the
Intensity Calibration.
The Calibration window closes.
Calibration window again.
15. Select the Use ILUT in Scope check box.
16. If you are using the original alignment plate supplied with the
instrument, select the Use Legacy Plastic Plate checkbox. This plate
has lined paper glued to the bottom.
The result should be a flat intensity readout (Figure H-4). If it is
not flat, repeat the ILUT calibration in step 4 through step .
Figure H-4 Calibrated intensity of Channels 1 and 2 with Laser 1
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169
method..
19. Click Apply n Quit at the bottom of the Calibration window.
The Calibration window closes and the new calibration values
are registered.
20. In the menu bar, click File > Save Method As and save the method
using the same file name.
The system is now calibrated for intensity with Laser 1.
Testing the ILUT Correction in Channels 1 and 2
After performing the ILUT procedure in Intensity Calibration (ILUT) for
Laser 1 (typically 488 nm) on page 166, calibrate and verify that these
regions are equivalent by visual inspection of the correction in the
Calibration window. After this is done, the ILUT correction can be
verified by making uniformity measurements using the Standard
Uniformity Plate (Paint Plate).
1. Use 510–540 nm filters are in Channels 1 and 2, use the
3. Open the ImageXpress Velos Software from the desktop
shortcut.
plate nest.
5. Locate the Standard Uniformity Plate (Paint Plate) and inspect
the bottom for scratches, marks, or debris. If acceptable, place
it into the plate nest with the plate’s A1 position in the top-left
corner.
6. Open Method for Uniformity.
7. If this is the first time running the Method, see Creating a
Method for the Standard Uniformity Plate on page 174.
Calibration window.
selected.
11. Select the Use ILUT in Scope check box.
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Figure H-5 Standard Uniformity Plate scan using channel 1 and
channel 2 in the calibration screen.
The alignment plate should show a flat correction down the
column in both channels from the cal plate ILUT calibration. The
focus should be between –100 μm and +100 μm of the 384
Matrical focus (0 and 0 for collection focus and scan focus).
If the signal is not uniform in intensity, either the ILUT was
incorrectly performed or the alignment plate is defective, for
example, it is tilted or warped.
13. Click the Stop button in the top-left corner of the window.
14. Close the Calibration window without applying any changes.
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171
Testing ILUT from Calibration
1. Use 510–540 nm filters are in Channels 1 and 2, use the
3. Open the ImageXpress Velos Software from the desktop
shortcut.
plate nest.
5. Locate the Standard Uniformity Plate (Paint Plate) and inspect
the bottom for scratches, marks, or debris. If acceptable, place
it into the plate nest with the plate’s A1 position in the top-left
corner.
6. Open Method for uniformity.
7. If this is the first time running the Method, see Creating a
Method for the Standard Uniformity Plate on page 174.
Calibration window.
selected.
12. Optimize the uniformity by adjusting the Scan Focus or
Collection Focus to get the best uniformity. After making minor
adjustments in collection focus if necessary to optimize the
uniformity, the focus values will usually be within 200 μm of the
zero setting.
13. Use a process similar to Test Process on page 173, with an ROI
which encompasses most of the well, anywhere from
1000x1000 to 2200x2200 rectangular. For more accurate
uniformity results, avoid the non-uniformity of the paint in the
edges of the well.
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Test Process
Create a process using the following parameters:
 Percent Background=0
 Threshold Low=1
 Threshold High=65535
 Area Filter=800 to 1E8
 Display=Channel 1
 Analyze Particles in=Channel 1
 Formula=None
 Smoothing=1
 Split=unchecked
 Flattening=0
 Show Flattening=unchecked
 Perimeter=do not use
 Mean Intensity=do not use
 Peak Intensity=do not use
 Integrated Intensity=do not use
 Anisotropy-1=do not use
 Anisotropy-2=do not use
 Region of Interest=2000X2000 rectangular for 384 wells
14. Acquire data and analyze with one of the 384 macros. Analyze
for CV of uniformity. For the entire plate this should nominally
be below 5%CV for each channel.
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173
Creating a Method for the Standard Uniformity
Plate
If the Method for the Standard Uniformity Plate (Paint Plate) is not
present, create a method with the following settings:
• Plate Type: 384 Matrical
• Scan Area: 3200 x 3200 μm
• Scan resolution: 10x10 μm
• Data Averaging: 1 avgs/pixel
Sensitivity 1X
Sensitivity 1X
• Channel 3: Enabled, Orange All, Gain between 500–700 mV,
Sensitivity 1X
• Channel 4: Enabled, Orange All, Gain between 500–700 mV,
Sensitivity 1X
• Method’s Collection Focus: –100 μm to +100 μm
• Method's Scanning Focus: –100 μm to +100 μm
• Image Analysis: Not Enabled
• Produce data (.bbd) file: Enabled
• Auto. Correct Saturated Data: Enabled
• Leave all other choices for analysis unselected
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1. Make sure that the filters in Channel 1 and 2, and the dichroic
mirror match the wavelength of Laser 2 (see Table H-1).
Table H-1 Matching laser & dichroic mirror
Laser Ch1 (nm) Ch2 (nm) Dichroic Mirror
(nm)
(nm)
2.
3.
4.
5.
6.
7.
8.
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405
430–480
430–480
495
440
460–500
460–500
510
532
560–610
560–610
610
640
690–800*
690–800*
690
*For general acquisition, the filter setting for the 640 nm laser is
660-800 for channels 1 and 2. For calibration, however, the
setting is 690-800 for channels 1 and 2.
Open Method for Alignment Plate.
From the menu bar, click View > Instrument Setup.
In the Instrument Setup dialog, click the Detect tab.
Click Laser 2.
Click OK.
From the menu bar, click Calibration > Calibration to open the
Calibration window.
The Laser 2 wavelength appears on the Ilut Cal button.
Follow Calibration procedure used for Laser 1. See Intensity
Calibration (ILUT) for Laser 1 (typically 488 nm) on page 166.
175
176
0270-00007 D
Creating a Plate Configuration File
I
• Preparation on page 177
• Creating or Modifying a Plate File on page 178
Preparation
1. Turn on the instrument and warm up 488 nm laser for at least
one hour.
2. Place the 510–540 nm (fluorescein) emission filter into
Channel 1 and the 488–10 nm (scatter) emission filter into
Channel 2. Use the 560 nm dichroic in both sides.
3. If possible, before starting the procedure, verify correct
calibration of the image. See ImageXpress Velos Image
Registration Alignment Procedure on page 137.
4. Pipet a 1:2000 dilution of 10 μm fluorescent beads into wells in
the first, middle, and last columns in the “plate of interest” or in
spots on the four corners of glass slide.
5. Centrifuge the plate for 2 minutes or allow beads to settle on the
glass slide for a few minutes.
6. Start the ImageXpress® Velos Software from the desktop
shortcut.
plate nest.
8. Place the “plate of interest” in the plate nest with the plate’s A1
position in the top-left corner.
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177
Creating or Modifying a Plate File
To create or modify a plate file, go through the following procedures in
the order given:
• To set up the plate file: on page 178
• To define a new method for the plate file: on page 180
• To set focus on the well bottom: on page 181
• To transfer the motor positions to the plate file: on page 184
• To check the A1 well alignment: on page 185
• To check the well-to-well spacing: on page 186
To set up the plate file:
1. Close any open methods.
2. From the ImageXpress Velos Software menu bar, click View >
Plate Configuration Editor.
3. In the PlateConfig dialog, click the Browse button
the right of the File name field.
4. Browse to: C:\Program Files\Molecular
Devices\ImageXpress Velos\cfg.
located to
Figure I-1 Plate configuration editor
5. Select the PlateTemplate.pt file, if you are creating a new plate
file.
The dimensions from the plate file populate the fields. For the
PlateTemplate.pt file, standard 384-well plate dimensions are
used. For other plates in the library, the dimensions were
originally determined by starting with the vendor-supplied
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measurements (converted to μm if given in mm) and then
optimizing empirically.
6. Select the file name at the far right side of the path statement in
the File name field. See Figure I-2.
Figure I-2 Editing the plate file name
7. Type a new plate name to replace the selected text.
The name you enter will be the name that appears in the
ImageXpress Velos Software plate menu.
8. Click Save.
9. In the Plate Dimensions fields, type the plate manufacturer’s
specifications in microns.
To preview the dimensions of the plate, click Draw Plate.
To restore the dimensions from the last time you saved, click
Retrieve.
10. Click Save.
Note: If the Windows directory is set to Read Only, you need
change the directory properties in Windows Explorer before you
can save the file.
11. Click Close to close the PlateConfig dialog.
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179
To define a new method for the plate file:
1. From the menu bar, click File > New to create a new method.
2. In the Instrument Setup dialog, choose the plate type you just
saved, and that matches the plate currently inside the
instrument.
Figure I-3 Choose a plate type
3.
4.
5.
6.
Click the Detect tab.
Select the Channel 1 and Channel 2 check boxes.
Clear the Channel 3 and Channel 4 check boxes.
In the Filter fields for each enabled channel, type descriptions of
filters used.
7. From the Polarization list for Channel 2, select Scatter.
180
0270-00007 D
Figure I-4 Enable Channels 1 and 2
8. Click OK.
To set focus on the well bottom:
Calibration window.
2. In the Scope Window area, verify that Display Channel 1 and 2
are selected.
3. In the Go to column field, type the number for a column in the
middle of the plate than contains fluorescent material, and then
click Set.
Note: It is best to determine focus using a column in the middle
of the plate rather than a column near a plate edge in case the
plate is not completely flat.
5. If intensity peaks are visible, adjust the Collection Focus up or
down in increments of 100 μm steps, until the peaks are at
maximum intensity. Stop at maximum intensity.
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181
6. Adjust the Scanning Focus the same distance.
For example, if you started at focus of 0, 0 and decreased the
collection focus by 100 μm, the Delta and Position of Motors will
be –100 μm. Make sure that the Scan Focus is also adjusted to
–100 μm.
7. Adjust the PMT gain for Channel 1 and Channel 2 so that the
highest peaks are no more than about 75% of the scope
window. Do not worry if Channel 2 with the scatter filter has one
or two “outlier” peaks that go off the top of the screen.
Figure I-5 Calibration window
method.
9. Close the Calibration window.
10. In the Plate Map window, select the center column and then
click the Scan button in the top-left corner of the window.
11. Double-click some wells in Channel 1 and Channel 2 to open the
Image window for those wells.
12. Visually determine if beads appear in focus. Enlarge and zoom in
on a well to look for uniform bead images.
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0270-00007 D
Figure I-6 Beads in focus
Beads in focus should appear fairly square. If rectangular, the
population should randomly include “portrait” and “landscape”
shapes.
If all of the beads are elongated in the same orientation or “V”
shapes are obvious, open the Calibration window (discarding the
data) and change the focus settings.
13. After the focus settings are optimized, follow the procedure To
transfer the motor positions to the plate file: on page 184.
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183
To transfer the motor positions to the plate file:
Calibration window.
2. In the Motors Jogging area, note the values in the Position
column for the Collection Focus and Scanning Focus fields.
Figure I-7 Collection Focus and Scanning Focus settings
Be sure to use the absolute Position, and not the Delta that is
also shown.
3. Close the Calibration window.
4. Close the method.
5. From the menu bar, click View > Plate Configuration Editor.
located to
8. Select the plate file you previously saved, and that matches the
plate currently inside the instrument.
9. In the PlateConfig dialog, type the Position values in the
Collection Focus and Scan Focus fields.
For example, if you adjusted the Collection Focus and Scanning
Focus to –800 μm, type –800 in the PlateConfig dialog.
10. Click Save.
11. Click Close to close the PlateConfig dialog.
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0270-00007 D
To check the A1 well alignment:
1. Open the method you created in To define a new method for the
plate file: on page 180.
2. From the menu bar, click View > Instrument Setup.
If a message appears asking if you want to discard your data,
click Yes.
instrument.
You should see the plate map “ripple” if changes were made.
4. Click OK.
6. Double-click well A1 to open the A1 Image window. If it is
difficult to see the edges of the well, use the brightness and
contrast controls on the toolbar to increase viewing brightness.
7. If the image is well centered, follow the procedure To check the
well-to-well spacing: on page 186.
If the image is too far right or too far left, then follow the
procedure in To center the A1 image left to right: on page 189.
Before
After
Figure I-8 Well A1 was too far right. It was centered by
decreasing the A1 Column Offset by 60 μm.
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185
If the image is too far up or too far down, then follow the
procedure in To center the A1 image top to bottom: on
page 190.
Before
After
Figure I-9 Well A1 was too far up. It was centered by increasing
the A1 Row Offset by 50 μm.
To check the well-to-well spacing:
1. From the menu bar, click View > Instrument Setup.
If a message appears asking if you want to discard your data,
click Yes.
instrument.
You should see the plate map “ripple” if changes were made.
3. Click OK.
6. Double-click bottom well in column 1 to open the Image window
for that well. If it is difficult to see the edges of the well, use the
brightness and contrast controls on the toolbar to increase
viewing brightness.
186
0270-00007 D
7. If both wells are centered, continue with step 8.
If the bottom well is higher or lower in the window than well A1,
then follow the procedure in To adjust the well spacing between
rows: on page 191.
Before
After
Figure I-10 Well P1 was too far down. It was centered by
decreasing the Row Spacing by 5 μm.
8. Close the Image window for the bottom well.
9. Double-click farthest-right well in row 1 to open the Image
window for that well.
10. If both wells are centered, continue with step 11.
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187
If the right well is farther to the left or right in the window than
well A1, then follow the procedure in To adjust the well spacing
between columns: on page 192.
Before
After
Figure I-11 Well A24 was too far right. It was centered by
decreasing the Column Spacing by 5 μm.
11. Close the image windows.
The calibration is complete
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0270-00007 D
Adjusting the Well Alignment
Use the following procedure to make adjustments to the well
alignment.
• To center the A1 image left to right: on page 189
• To center the A1 image top to bottom: on page 190
• To adjust the well spacing between rows: on page 191
• To adjust the well spacing between columns: on page 192
To center the A1 image left to right:
located to
6. In the PlateConfig dialog, type a new value in the A1 Column
Offset field.
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189
Figure I-12 Editing the A1 Column Offset field
If the image was too far left in the Image window, increase
the value in A1 Column Offset field.
 If the image was too far right in the Image window, decrease
the value in A1 Column Offset field.
7. Click Save.
8. Repeat the procedure To check the A1 well alignment: on
page 185 until A1 is well centered from left to right.

To center the A1 image top to bottom:
located to
6. In the PlateConfig dialog, type a new value in the A1 Row Offset
field.
190
0270-00007 D
Figure I-13 Editing the A1 Row Offset field
If the image was too far up in the Image window, increase
the value in A1 Row Offset field.
 If the image was too far down in the Image window,
decrease the value in A1 Row Offset field.
7. Click Save.
8. Repeat the procedure To check the A1 well alignment: on
page 185 until A1 is well centered from top to bottom.

To adjust the well spacing between rows:
located to
6. In the PlateConfig dialog, type a new value in the Row Spacing
field.
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191
Figure I-14 Editing the Row Spacing field
If the image was too low in the Image window, increase the
value in Row Spacing field.
 If the image was too high in the Image window, decrease the
value in Row Spacing field.
7. Click Save.
8. Repeat the procedure To check the well-to-well spacing: on
page 186 until both wells are well centered from left to right.

To adjust the well spacing between columns:
located to
6. In the PlateConfig dialog, type a new value in the Column
Spacing field.
192
0270-00007 D
Figure I-15 Editing the Column Spacing field
If the image was too far right in the Image window, increase
the value in Column Spacing field.
 If the image was too far left in the Image window, decrease
the value in Column Spacing field.
7. Click Save.
8. Repeat the procedure To check the well-to-well spacing: on
page 186 until both wells are well centered from top to bottom.

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193
194
0270-00007 D
Maintenance
J
• Cleaning the Optics on page 195
• Transfer Data on page 196
To clean the optics, use a clean pressurized air source that removes
dust particles without depositing oil or any other material on the lenses
and mirror. For example, Dust-Off® compressed air duster is ideal for
removing airborne particles from optics. This gas propellant uses an
environmentally safe formula that incorporates a hydrogen atom which
allows it to decompose before it reaches the atmosphere.
CAUTION! Do not use the facilities compressed air source to clean the
optics as these sources usually contain oil droplets or other particulate
matter that can coat the optics.
Cleaning the Optics
CAUTION! Before you begin the cleaning procedure, turn off the system
power to ensure that the laser and exposed internal electrical lines are
not powered during the maintenance procedure.
1. Grasp the handle of the emission filter compartment and tilt the
cover out and down, away from the instrument.
2. Pull the knob out slightly and move the inner door to the right to
expose the filters for channels 1 and 3 or to the left to expose
the filters for channels 2 and 4.
3. Remove each filter and dichroic mirror by pulling it straight out
by the knurled knob.
4. Use a clean compressed air duster to remove any particles from
each filter.
5. Inspect both sides of each filter for signs of “freckling”,
bleaching, or delamination of the filter layers.
6. If filters appear damaged or excessively bleached, replace with
new ones.
7. Replace filters in correct positions.
8. Slide light-tight cover to the center/closed position and shut the
compartment cover.
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195
Maintenance
Transfer Data
Periodically transfer data from the computer hard drive to avoid
problems that can be caused by low disk space.
196
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Index
A
cytometer
connecting to computer 23–25
filters 101
how it operates 20
overview 19
adaptive intensity threshold 64
analysis processing control panel 61
threshold parameters 64
analysis results 95
(.bba) 112
particle summary 78–79
anisotropy 21, 29
data acquisition quick start guide
anisotropy factors 66
101–103
auto-analysis mode 91–93
autorun mode 121–133
simulated robot 128–133
start, pause, or abort 134
Twister II microplate handler
122–127
electrical safety 14
emission filters
change 28
verify 27–29
D
E
B
barcode 123
brightness 59–60
C
F
FCS compatible file 113
FCS Express compatible file 113
Calibration control panel
Channel adjustments 52
calibration mode 37–53
cleaning optics 195
collection focus 48
connecting computer to cytometer
23–25
contrast 59–60
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197
Index
file types
analysis results (.bba) 112
FCS compatible 113
FCS Express compatible 113
image method (.bbd) 111
MDCStore compatible 113
Method derived from ___ (.bbd)
111
plate analysis results (.plate.txt)
111
post-process (.ppr) 112
process (.bbp) 112
well analysis results (.csv) 111
filters 101, 115–117
change 28
verify 27–29
fixed intensity threshold 63
flattening 65
formula 65
H
hazardous material precautions 15
high-voltage hazard 12
L
laser
class & power 12
optics 21
laser safety 12–14
laser scanner
optics 21
lid 123
lifting hazard 11
loading a microplate 29
M
maintenance 195
MDCStore compatible file 113
mechanical safety 15
method
create 31–40
open 41
Method derived from ___ (.bbd) 111
microplate
configuration 178
loading 29
I
image data (.bbd)
export 90–??
open 56
save 88
image method (.bbd) 111
image registration alignment 137,
153
intensity calibration 165–175
intensity threshold 62
adaptive 64
fixed 63
198
N
nest 123
O
open
image data (.bbd) 89
method 41
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optics 21
maintenance 195
optimize scanner settings 37–53
overview 19
S
safety
general 11
safety information 10–15
electrical 14
general safety 11
hazardous material precautions
15
particle filters 67
high-voltage hazard 12
particle summary 78–79
laser 12–14
plate analysis results (.plate.txt) 97,
lifting hazard 11
111
mechanical 15
plate configuration 178
safety labels 16
polarization filters
warning labels 17
verify 27–29
safety
labels 16
post-process (.ppr) 112
save
process
image data 88
create 55–74
processed data 96
threshold parameters 64
scan
a plate 86–??
process (.bbp) 112
scan
line position 46
processed well data 75–76
scan time 119
scanner
See laser scanner.
scanning focus 48
signal 42
adjust amplitude 51
quick start guide for data acquisition simulated robot autorun 128–133
101–103
smoothing 65
software
quick start guide 101–103
starting 26
specifications, system
start software 26
raw data (well image) 87
support 18
real-time signal 42
system
specifications 22–23
resolution 119
ROI 57–59
P
Q
R
0270-00007 D
199
Index
T
technical support 18
toolbar 105–110
V
validated fluorophores 115–117
verify filters 27–29
view
analysis results 78
processed well data 75–76
W
warning labels 17
well
analysis results (.csv) 111
image (raw data) 87
results (.csv) 99
200
0270-00007 D

ImageXpress Velos Laser Scanning Cytometer User Guide

Transcription

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