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					Microarray Scanner Start-Up Kit
            ArrayVIEW Demo

   Synapse Strategic Product Development LLC.
Table of Contents

Introduction……………………………………………………………………………......4
    What is a Microarray?……………………………………..………………………......4
    What is a Microarray Scanner?………………………………………..……………....4
    What is ArrayVIEW?………………………….………………………..…………......4
     Using this document………………………………………………………………......4
Part I, Array VIEW User Manual User……………………………………………………5
    ArrayVIEW User Manual…………………………………………………..…………6
     Getting Started……………………………………………………..………………….6
    Installation…………………………………………………...………………..…….…6
    Uninstall…………………………………………………………………………..…...6
System Requirements……………………………………………………………………...7
     PC Requirements………………………….………………………………………..…7
    Supported Motion Hardware……………………………………………………..……7
    Vision……………………………………………………………………………..…...7
Using the Microarray Start-up Kit………………………………………………………...8
     Navigating the Front Panel………………………………………………………..…..8
    Scan……………………………………………………………………………..……..9
    AutofocusDemo………………………...……………………………………..……..11
    Analysis Demo………………………………………………………………..……...12
    Motion Setup…………………………………………………………………..……..13
     Camera Settings and Filters…………………………………………………..……...14
Part II, Array VIEW Design Discussion…………………………………………………16
    System Overview…………………………………………….……………….……...17
    Software…………………………………….……………………………….….……17
     Motion Software………………………………………………………………..……17
     Vision Software………………………………………………………………..…….18
Optics…………………………………………………………………………………….19
     Camera……….……………………………………………………………………...19
     Objective Lens…………………………………………………….………………...20
     Field Lens…………………………………………………….……………………...20
    Optics Calculations………………………………………………………..…………20
    Auto focus………………………………………………………………..…………..22
Fluorescence Microscopy………………………………………………………………..23
     Olympus Microscopy Resource Center…..………………………………………….23
     Filter Cube Turret…………………………..………………………………………..23
    Filter Cubes………………………………..…………………………………………23
     Illuminator…………………………….……………………………………………..23
Sample Handling…………………………………………………………………………24
     Motion Control..……………………………………………………………………..24
     Chassis………………………………………………………..……………………...25
Conclusion……………………………………………………………………………….25
 Appendix A, Bill of Materials…………………………………………………………...26




                              2
Disclaimer
This application is for demonstration purposes only. Use this software at your own risk.
Synapse is not responsible for any damage to you or your equipment due to using this
software. If you need for information on developing a custom imaging platform please,
contact synapse.

Synapse Product Development
3406 East Union Street
Seattle, WA 98122

Phone: 206-381-0898
info@synapsedev.com




                                            3
Introduction

What is a Microarray?

A Microarray is an array of very small test sites, each of which carries out a specific
experiment. You may array thousands of test sites onto a single test slide, thus greatly
simplifying and expediting the testing process. With this increase in testing capability
comes the burden of increased data handling and a requirement for an automated method
with which to handle this data.


What is a Microarray Scanner?

A microarray scanner collects data from a microarray in an automated fashion. This
device is essentially an automated microscope with special software designed to ease the
task of image quantification and data analysis.


What is ArrayVIEW?

ArrayVIEW is a low-cost microarray scanner made from National Instruments hardware
and software. ArrayVIEW combines proven off-the-shelf optical technologies with
flexible sample handling and data analysis.


Using This Document
This document is divided into two parts.

Part I contains the User’s Manual for ArrayVIEW and familiarizes the user with the
functions of the ArrayVIEW application.

Part II contains an in-depth discussion on the implementation of a microarray scanner
using National Instruments hardware and software. It includes details regarding
component selection and sourcing, as well as some critical information to consider in the
design process.




                                            4
  Part I.

ArrayVIEW
User Manual




     5
ArrayVIEW User Manual
This version of ArrayVIEW is a demonstration application designed to show how you
can use National Instruments hardware and software to produce a custom microarray
scanner. This application is fully functional, but also will operate in demo mode without
the proper hardware.

Getting Started
ArrayVIEW requires that certain software applications and drivers be loaded prior to
running. The order of installation is important; please follow the installation instructions
carefully. The associated hardware need not be present to explore the primary features of
the application.

ArrayVIEW requires
To install ArrayVIEW [redpr, the following functions:
   1. Download MicroArray Start-Up Kit.zip to your target PC
   2. Expand the zip file (the following instructions assume the package was expanded
       in C:\Temp)
   3. Locate and run QCapSuite260.exe. This application installs windows drivers for
       the camera used by ArrayVIEW
   4. Locate and run the LabView100.exe. This application installs the LabVIEW
       drivers from QImaging
   5. Locate and run “ArrayVIEW Install”

You are now ready to run the ArrayVIEW application, and you may select it from the
Windows Start Menu.

Uninstall
To uninstall ArrayVIEW, perform the following actions:

   1.   Navigate to the Windows Control Panel
   2.   Select “Add/Remove Programs”
   3.   Select “ArrayVIEW”
   4.   Proceed with uninstall
   5.   In “Add Remove Programs, select “Q Imaging LabVIEW Interface”
   6.   Proceed with uninstall
   7.   In “Add/Remove Programs, select “Q Capture Suite”
   8.   Proceed with Uninstall




                                             6
System Requirements
ArrayVIEW performs mathematically intensive functions during the image processing.
If the minimum system requirements are not met, degraded performance may result. The
minimum system hardware for ArrayVIEW is shown below.


PC Requirements
           -   Processor: 1GHz Pentium 4
           -   Minimum suggested RAM: 512 MB
           -   Free hard drive space: 40 Mb
           -   Windows 9x, Windows 2000, Windows XP
           -   IEEE 1394 Firewire Port (Require only to use the Camera)



Supported Motion Hardware
Utilizing ArrayVIEW’s motion functions require the following hardware:
           - Controllers Supported
               NI 7334 low-cost motion controller
               NI 7332 low-cost motion controller (no third axis for autofocus)
               NI 7344 high-performance motion controller
               NI 7342 high-performance motion controller (no third axis for autofocus)

           - Stepper Motor Drives Supported
             NI 7604 stepper motor drive
             NI 7602 stepper motor drive (no third axis for autofocus)

Vision
Utilizing ArrayVIEW’s image capture functions requires the following hardware:
           - Camera
               Q Imaging Retiga 1300C (cooled CCD camera)
                     or
               Q Imaging Retiga 1300

           - IMAQ Vision Runtime and License
           - Available IEEE-1394 Firewire Port




                                           7
Using the Microarray Start-Up Kit

Navigating the Front Panel
The front panel of ArrayVIEW has a set of tabs that control different features of the
application. Some tabs contain demos and others save you the ability to configure
ArrayVIEW for your hardware. The features of these tabs are described in detail in the
following sections.




                          Figure 1: ArrayVIEW Front Panel




                                           8
Scan
The Scan tab demonstrates the main functions of a microarray scanner. Pressing the
“Start” button begins the demo or activates the imaging sequence if you have enabled
your hardware. In demo mode, the microarray scanner simulates a scan of a microarray.




  Scan setup for
  hardware

  Main image
  window

  Scrolling Status
  window shows
  the current
  action

  Stitched images
  of the
  microarray




                                    Figure 2: Scan Tab

We took images used for this demo on a custom microarray scanner built by Synapse. If
you have enabled the hardware, ArrayVIEW takes control of your hardware and performs
the microarray imaging routine defined by the user settings.

Scanning a microarray is a complicated task and watching the demo may be confusing
the first time. As the demo runs, the software performs a series of actions. These
actions are outlined below along with the icons used to represent the hardware involved.

Autofocus: The Autofocus demo uses prerecorded images to demonstrate
a LabVIEW implementation of a contrast-based autofocus system.

Motion: The samples are positioned under the camera with an automated
XY stage. The stage allows the scanner to scan multiple samples and
mechanically stitch the images together for larger samples. For simplicity,
the demo application can only be set up to scan a simple sample.

Automated Filter Changes: The Olympus BX-RFAA Motorized Filter
Cube Turret makes automating filter cube changes a snap. The demo uses
the CY3 and CY5 filters to image a calibration slide.


                                             9
Imaging: The images shown in ArrayVIEW were captured using a cooled CCD
camera made by Q Imaging Corp. This camera captures 12-bit monochrome
images. Filter cubes are used to image the microarray in multiple wavelengths.
For this demo, the images are displayed in false color for easy identification.

2D Stitching: You can use stitching when samples are too large for one
image at the desired magnification. For demonstration purposes, we have
stitched a 2x2 image.



The user settings on the Scan Engine tab are only used if hardware has been enabled.
The purpose of these buttons is outlined below.




        Enable Motion: This button activates your motion hardware. You can drive
        motion without a camera enabled. The control is shown with motion hardware
        enabled.
        Enable Camera: This button activates your vision hardware. You can use your
        camera without motion enabled. The control is shown with the camera enabled.
        Sample Name: This is the name you use when saving the images taken by
        ArrayVIEW.
        Directory: This is the directory that your images will be saved to.
        Filter Selection: You can select each filter you would like to use in a scan.




                                              10
Autofocus Demo
This tab contains an interactive demo of an Autofocus algorithm. The graph shows the
image contrast over a range of focus. Pressing the "Demo" button starts the algorithm.




Autofocus images of
a silicon die are
prerecored.


This plot depicts
contrast vs the
position of the
objective lens.


Press the run button
to see the Autofocus
demo.

                                Figure 3: Autofocus Tab

This demo is using prerecorded images and real-time image processing. This simulates
an objective lens moving through a range of motion. An image processing algorithm is
used to compare the contrast values of the image. The location with the highest contrast
represents the point of best focus, and this position is recorded at the end of the move.
The lens is then repositioned to the location with highest contrast and best focus.




                                            11
Analysis Demo
Taking an image of a microarray is only half the battle. Extracting the data can be a time
consuming process that involves manually defining the site locations or the use of
expensive software. National Instruments IMAQ vision package gives you the tools to
create a custom detection routine for your microarray. This demo masks background
noise and quantifies the signal associated with each site. The Quantify Array algorithm
calculates the mean intensity of each site and organizes the values in an array.




Original misaligned
image from the
imager


Location of the original
image relative to the
known pattern



 Quantified
 intensities from the
 microarray




                                  Figure 4: Analysis Demo Tab

        Find Array: The “Find Array” button starts an algorithm that maps the
        microarray within the sample image. This demo searches for the expected array
        pattern and outputs location and orientation. It measures the returned offset in
        pixels from the top left corner and the angle in degrees.
        Quantify Array: The “Quantify Array” button starts an algorithm that masks out
        background noise and quantifies the signal associated with each site. The
        Quantify Array algorithm calculates the mean intensity of each site and organizes
        the values in an array.




                                            12
Motion Setup
You use the Motion Setup tab to configure the motion hardware and define the sample
location and size. You use these settings only when hardware is present. The diagram on
the right shows the general layout, size, and shape of the sample. For simplicity this
demo only allows one sample to be set up. The settings are described below.




Motion setup and
sample layout
controls


Diagram shows a
graphical
representation of the
sample layout


Adjust the scaling
factor to keep your
layout on the page


                              Figure 5: Motion Setup Tab

       Motion Board: Set the Board ID for your motion controller card. You can find
       it in the Automation Explorer. Set the Maximum Velocity and acceleration
       values for your system. Test these values before you run the system to prevent
       damage.
       Stitch Sizes: Calculate the number of steps equal to the height and width of your
       camera’s field of view. This will be the steps per stitch, where x is the width and
       y the height. You must use integer values.
       Sample Location: This is the top left corner of your sample, and all stitching
       will be done relative to this corner in steps from the origin (0,0). You must use
       integer values.
       Number of Stitches: This is the number of images you would like to stitch
       together to form a composite of your sample. You must use integer values.
       Layout Diagram: This gives a graphical representation of the sample layout.
       The Origin is in the bottom left corner. You can adjust the scaling factor to fit
       your layout in the diagram. Ignore this diagram if your system has a different
       layout.



                                            13
Camera Settings and Filters:
You use this tab is used to adjust the camera settings for each filter used during a scan.
You can verify the settings using the Current Filter selector and Preview Image button.

Each filter set allows specific frequencies and intensities of light to reach the camera.
This makes it necessary to configure the camera for each filter individually. The
ArrayVIEW application supports up to six filters and their corresponding camera settings.
Each filter has a control on the “Camera Settings and Filters” tab. These settings are
described below.




Test camera settings
usin the treview
window

Each filter has its
own camera
settings.

Set the filter you
want to preview and
press the preview
button to capture an
image.
                              Figure 6: Camera Settings Tab

       Binning:
       Integer values of one, two and four select the image size taken by the camera.
       Binning of one is the full size to the CCD array. Binning of two cuts the image in
       half and four takes it to one quarter of its original resolution. With binning the
       camera can use shorter exposure times at the sacrifice of resolution.

       Offset:
       With this setting you can apply a constant offset to the resulting image data. The
       default setting is 1680.
       Gain:
       With this setting you can apply a multiplication factor to the resulting image data.
       The default setting is 1280.




                                             14
Exposure:
The exposure time is the time CCD is allowed to collect light. The value is
measured in microseconds. An exposure value of 1,000,000 is equivalent to 1
second. It is important to set the exposure separately for each filter because each
passes different amounts of light. You can set the exposure to a maximum of 16
seconds for the Q Imaging Camera. This setting must be an integer value.




                                     15
    Part II.

  Array VIEW
Design Discussion




       16
System Overview
The ArrayVIEW scanner consists of three major components: software, sample handling,
and optics. This section discusses in some detail and where appropriate, provides
specific information regarding the design of the ArrayVIEW microarray scanner.




                                  Software


                                    Optics


                           Sample Handling



Software
Developing software for a custom microarray scanner can be a daunting task. However,
using National Instruments LabVIEW in conjunction with motion and IMAQ Vision
products can greatly decrease your development effort.

ArrayVIEW is a fully functional microarray scanning application that can perform a
variety of tasks to help image and quantify a microarray. We wrote this application
completely in LabVIEW and it maintains a high level of functionality and robustness.

The system software can carry out three primary tasks: image manipulation, image
management, and motion control.

Motion Software
National Instruments FlexMotion software is a proven software package that you can use
to issue high level commands to execute desired motion operations. You can easily
implement the motion software requirements of the microarray scanner using this
package.

The ArrayVIEW scanner uses two axes of the motion controller for sample handling and
a third for autofocus. Additionally, you can use the basic DIO functionality of the motion
controller to drive a custom interface to an automated filter cube turret.


                                           17
Vision Software
Due to the relatively large size of a microarray, the limited field of view, and the high
resolution required for a microarray image, the final image must be a composite of
multiple images taken individually. IMAQ Vision contains a wealth of image processing
functions that are robust and well implemented. Using the IMAQ image-to-image and
copy functions, you can assemble individual tiles into a complete mosaic of the original
microarray.

The autofocus and microarray analysis portions of the ArrayVIEW application use
additional IMAQ Vision functions such as contrast detection and pattern matching.

Note: Take care when manipulating a 12-bit grayscale image in IMAQ Vision or any
other image processing routine. Image data is often cast into an 8-bit image without any
apparent effects. However, this results in a 16x decrease in overall sensitivity of the
instrument. Boundary checking on the final output image ensures that no unnecessary
loss of data has occurred.




                                           18
Optics
Custom optical component design is expensive and time consuming. ArrayVIEW greatly
simplifies this task by using off-the-shelf optical components from Olympus. The
objective lens, filter turret, and field lens are standard components of the Olympus BX-51
series microscopes.



                                  Camera


                                   Field Lens
                                                                   Lamp

                                       Filter
                                         Turret




                                       Autofocus
            Objective
             Lens

                         Figure 7: BX-51 Typical Optical Train


Camera
To quantify a microarray using CCD imaging technologies, you must use a high-quality
cooled CCD camera. We used the Retiga 1300C cooled CCD camera from Q Imaging
Corporation was used to capture the images shown in the ArrayVIEW demo. This
camera has a resolution of 1280 x 1024 pixels with 12 bits per pixel grayscale.




                           Figure 8: Q Imaging Retiga 1300C


                                           19
The Retiga 1300C has a Firewire interface and a custom LabVIEW driver that may be
obtained directly from Q Imaging.

Objective Lens
The objective lens used in the ArrayVIEW scanner is a 4x U-Plan Flourite lens with a
numerical aperture of 0.13 and a working depth of 17nm, Olympus part number 1-
UB522. This lens is specifically designed for the wavelengths used in fluorescence
imaging.

Field Lens
To utilize a CCD camera with an Olympus optical system, you must use a field lens. This
lens captures the light from the objective lens and focuses the image on the plane of the
CCD. Olympus field lenses are designed to be used in conjunction with either the
standard microscope eyepiece or the field lens adapter. The lenses used in the
ArrayVIEW scanner are the U-TV0.5XC field lens and the U-TLU field lens adapter. In
combination with the 4x objective lens, this gives a total magnification of 2x.


Optics Calculations
When designing a microarray scanner, you should consider the required resolution.
Often expressed in microns per pixel, the resolution is a measurement of the smallest
feature size that an imaging system can resolve.

Using off-the-shelf optical products gives the designer the ability to use straightforward
calculations in order to determine the required system magnifications. For a microarray
scanner a good rule of thumb is that the microarray sites should be imaged with at least 5
pixels, and preferably 10. For example, if the minimum spot size of the microarray is 40
microns, then a minimum resolution of 8 microns is required and 4 microns is desirable.




                                            20
The calculations shown below assume that the required feature size is 4 microns. Using
this objective lens and the specified field lens gives a total system field of view of 4.29
mm by 3.43 mm.

                Camera Resolution
                           Horizontal Pixels                       1280    pixels
                           Vertical Pixels                         1024    pixels

                Pixel Size
                                Length                               6.7   µm
                                Width                                6.7   µm

                CCD Size
                                Horizontal Pixels x Pixel Width     8.58   mm
                                Vertical Pixels x Pixel Length      6.86   mm

                Magnification
                             Objective Lens                          4.0   X
                             Field Lens                              0.5   X

                Total Magnification
                             Objective Lens x Field Lens             2.0   X

                Field of View
                                CCD Width / Total Magnification     4.29   mm
                                CCD Length / Total Magnification    3.43   mm

                Resolution
                                Pixel Size / Total Magnification    3.35   µm/pixel

Should your system require a different field of view, the calculations shown above may
be helpful in determining the correct magnification.




                                                21
Autofocus
To ensure the images captured by the scanner are in focus, the maximum variation in the
distance from the objective lens and the sample must be no more than 15 microns.
Achieving this goal for more than one image at a time can be cumbersome; therefore an
automated focusing mechanism is necessary in order to successfully image a microarray.

Synapse has developed a low-cost autofocus mechanism that adapts easily to the standard
Olympus objective lens mount. This mechanism uses the scanner’s existing optical and
imaging hardware in addition to custom positioning hardware to extend the system depth
of focus to over 5 mm. The ArrayVIEW application includes a demo of this mechanism
in operation.




                     Figure 9:    Synapse Autofocus Mechanism
                       (Compatible with Olympus Microscopes)




                                          22
Fluorescence Microscopy
The following discussion assumes that the reader is familiar with fluorescence
microscopy. If you are unfamiliar with fluorescence and dichroic imaging, please visit
the following website for more information.


Olympus Microscopy Resource Center
       http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html


Filter Cube Turret
The Filter Cube Turret is a mechanical device used to hold multiple Filter Cubes. With
an automated filter cube turret such as the BX-RFAA from Olympus, you can use up to
six different filter cubes in fluorescence imaging.

Typically, control of an automated filter cube turret requires proprietary hardware and
software. To ease the task of integrating a filter cube turret into custom applications,
Synapse has created hardware and software that you can use to control the automated
filter cube turret from LabVIEW via DIO available on NI Motion Controllers.

Filter Cubes
We took the images shown in the ArrayVIEW demo using Cy-3 and Cy-5 fluorescence
tags through dichroic filter cubes. You can obtain the filter cubes used in the
ArrayVIEW scanner from Chroma Technologies, using the part numbers are shown
below.

                        Cy3
                                  540/25x             Exciter
                                  595/50m             Emitter
                                  Q565LP              Dichroic

                        Cy5
                                  635/30x             Exciter
                                  685/40m             Emitter
                                  Q660LP              Dichroic

Illuminator
The excitation source used in the ArrayVIEW scanner is a 75W Xenon burner using the
Olympus Lamphouse and Power Supply. Please see attached Bill of Materials for part
numbers.




                                            23
Sample Handling
The most flexible portion of the ArrayVIEW scanner is the sample handling. Its
flexibility comes from the use of National Instruments hardware and software products
for motion control. Using these products as a basis for the sample handling subsystem
allows for automated handling of multiple samples and various formfactors.

Motion Control
The Motion Control subsystem used in the ArrayVIEW scanner consists of an NI 7334
motion controller and an NI 7604 stepper motor drive. These components coupled with
off-the-shelf linear actuators and stepper motors give you the necessary mechanical
accuracy to create a mosaic of tiled images.




                  Figure 10:     Typical Sample Handling Mechanism

To ensure the tiled images can be taken correctly, several issues must be considered
during the component specification. Mainly, the mechanical resolution of the motion
subsystem must exceed the resolution of the optics subsystem. The following
calculations demonstrate the mechanical resolution of the components used in the
ArrayVIEW scanner.




                                           24
Mechanical Accuracy:

              Stepper Motor
                Degrees per Revolution                  A    360    deg
                Degrees per Step                        B     1.8   deg

                 Steps per Revolution (A/B)             C    200    steps

              Microstepping
                Microsteps per Step                     D     25    µsteps

              Actuator Lead Screw
                Millimeters / Revolution                E      2    mm

              Mechanical Resolution
                Millimeters / Step (E/C)                F   0.01    mm
                Micrometers / Microstep (F/D)                0.4    µm



It is important to note that the actual mechanical resolution will be somewhat less than
the calculations shown due to friction. It is therefore necessary to exceed the optical
resolution by some margin. For reference, it has been our experience that the above
mechanical system is sufficient to enable tiling of images taken with an optical resolution
of 4 µm per pixel.


Chassis
The chassis design of a microarray scanner can be rather simple, however care should be
taken to ensure that the overall stiffness of the structure be sufficient to prevent unwanted
vibrations during motion. These vibrations can have an adverse effect on the imaging
process and may require you to operate the motion system at less than optimum speed.


Conclusion
Using National Instruments hardware and software as the basis of a custom microarray
scanner is cost effective and efficient. Synapse Product Development has created
multiple imaging platforms and components that you can use as the core of your custom
imaging instrument.

Please feel free to contact Synapse for more information.

Synapse Product Development
3406 East Union Street
Seattle WA 98122

info@synapsedev.com



                                              25
                         Appendix A.
                         Bill of Materials

                        Item #   Qty.   MNFG                   Part #          Description
                          101     1     Generic                                PC
Control / Electronics




                          102     1     National Instruments   776670-03       Vision License
                          103     1     National Instruments   777935-01       PC-7334
                          104     1     National Instruments   777936-01       MID-7604, 4-Axis Stepper Driver and Breakout
                          106     2     National Instruments   186380-02       2 Meter Shielded Cable
                          106     1     Synapse                O-S001          Fluorescence Turret Interface
                          106     1     Synapse                F-S002          Autofocus Mechanism

                         200      1     Q Imaging              Retiga 1300C    1280x1024 IEEE1394 CCD Imager
                         201      1     Olympus                U-TV0.5XC       Field Lens
                         202      1     Olympus                U-TLU           Field Lens Adaptor
                         203      1     Olympus                BX-RFAA         Fluorescence Turret Illuminator 6-Cube
                         204      1     Olympus                1-UB522         U Plan Fluorite 4X, NA 0.13, WD 17mm
Optics




                         205      1     Olympus                U-LH7575XEAPO   Xenon Lamp Housing
                         206      1     Olympus                AH2-RX-T        Xenon Power Supply
                         207      1     Olympus                8-B198          75W Xenon Burner
                         208      1     Chroma Tech            41007           CY3 Filter Cube
                         209      1     Chroma Tech            41008           CY5 Filter Cube

                         300      2     Thomson                H42S344001      Size 23 Stepper
                         301      2     Thomson                MS33-LAB-L400   Mechanical Slide 300 mm
Mechanical




                         302      1     Thomson                MS33-LAB-L300   Mechanical Slide 200 mm
                         303      3     Thomson                LSP-MS33-3      Limit and Home Switches




                                                                        26

				
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