Transcript
INOVA Diagnostics, Inc.
NOVA View® Automated IFA Microscope Installation and Calibration Manual IMPORTANT: Please read carefully before using NOVA View® System
ECR Number: 427362 Effective date: 03/08/12 NOVA View Installation and Calibration Manual Rev.0
INOVA Diagnostics, Inc. 9900 Old Grove Road San Diego, CA 92131 United States of America Technical Service (U.S. & Canada Only):+1-877-829-4745 Technical Service (Outside the U.S.): +1-858-805-7950
[email protected] NOVA View® Automated IFA Microscope Release Information
INOVA reserves the right to make technical improvements to this equipment and documentation without prior notice as part of a continuous program of product development. This manual supersedes all previous editions. Information in this document is subject to change without notice. Copyright © 2011 INOVA Diagnostics, Inc. All rights reserved. This document is the copyright of INOVA, Inc. and must not be copied or reproduced in any form without prior consent.
NOVA View Installation and Calibration Manual Rev.0
NOVA View® Automated IFA Microscope
Installation and Calibration Manual
IMPORTANT: Please read carefully before using NOVA View® System
NOVA View Installation and Calibration Manual Rev.0
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NOVA View Installation and Calibration Manual Rev.0
Table of Contents
About This Manual ............................................................................................. 7 Installation and Calibration Overview ............................................................. 8 CHAPTER 1: Installation .................................................................................... 9 1.1 Unpacking the Instrument ................................................................................... 9 1.2 Setup of Main Hardware ..................................................................................... 13 1.3 Installation of Instrument Cables ....................................................................... 22
CHAPTER 2: Software Initiation ..................................................................... 35 CHAPTER 3: Centering the Optical Light Pathway ....................................... 39 CHAPTER 4: System Cover Installation .......................................................... 45 CHAPTER 5: Stage Calibration ........................................................................ 51 5.1 Stage Calibration – X & Y .................................................................................... 51 5.2 Stage Calibration – Z .......................................................................................... 56
CHAPTER 6: FITC Light Intensity Calibration ................................................ 63 CHAPTER 7: Backup of Important Files ......................................................... 67 7.1 Settings File ........................................................................................................ 67 7.2 PRO File .............................................................................................................. 68
CHAPTER 8: Performance Characteristics and Specifications ..................... 69 CHAPTER 9: Troubleshooting ......................................................................... 71 APPENDIX A: Instrument Cables .................................................................... 73 APPENDIX B: Checklist of Components Shipped with System ................... 75 APPENDIX C: Installation Record ................................................................... 77 APPENDIX D: Instructions for Software Installation and Upgrade ............. 79
NOVA View Installation and Calibration Manual Rev.0
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NOVA View Installation and Calibration Manual Rev.0
About This Manual This manual has been written for trained service personnel. It provides detailed instructions for setting up the NOVA View Automated IFA System. This manual gives you the information needed to:
Unpack the NOVA View components
Assemble the NOVA View system
Install the NOVA View system and software
Calibrate the NOVA View stage and optics
Calibrate the NOVA View intensity
Backup instrument files
Troubleshoot system errors
For instructions on system use and maintenance, please refer to the NOVA View Operator’s Manual.
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Installation and Calibration Overview
Remove the packaging and check for damage to any of the contents.
Verify that all materials in the packing list are present.
Install the base on a table or lab bench.
Position the main components on the base.
Connect component cables.
Power on system and initiate software.
Verify microscope optical settings.
Install system cover.
Set the working coordinates for the robotic stage to the inverted microscope (X, Y, and Z).
Calibrate the FITC light intensity and set the correction factor.
Backup important files.
Caution The installation and calibration must be executed exclusively by personnel who are adequately prepared and trained.
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Installation
CH. 1
CHAPTER 1: Installation The installation procedure includes the set up of the main components and instrument cable connections. Installation of the NOVA View system must be completed by an INOVA field service representative or a trained engineer. Deviations from this procedure can affect instrument performance and should be avoided. Documentation of the installation is recommended (see Appendix C: Installation Record).
1.1
Unpacking the Instrument
The NOVA View system is delivered in two boxes. The upper box contains the system base and cover. The lower box contains the system hardware components and cables. A backup power supply unit, printer, and other optional external components that may be provided will be shipped separately. The upper box contains the system cover and the base of the instrument.
Figure 1-1 Upper box containing system cover and base The lower box contains 3 different levels. The top level contains the monitor, power strip, keyboard, and fiber optic cables (stored beneath the keyboard). Depending on the crate configuration, the mirror unit may be stored beneath the keyboard or in its own compartment. The camera adaptor may be stored in its own compartment or in the middle box containing the instrument cables.
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Figure 1-2 Top level of lower box containing monitor, power strip, keyboard, fiber optic cables and mirror unit
Figure 1-3 Top level of lower box with designated compartments for the mirror unit and camera adapter The middle level is a box with several compartments containing the instrument cables, LED handheld unit, and mouse. The camera adapter and slide stage cover may also be shipped in one of these compartments.
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Figure 1-4 Middle level cable compartment box, as it fits in the lower box
Figure 1-5 Middle level cable compartment box The bottom level contains the microscope (with stage attached), microscope control unit, LED unit, camera, collimator, computer, monitor base, and tool kit.
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Figure 1-6 Bottom level containing microscope, microscope control unit, LED unit, camera, collimator, computer, monitor base, and tool kit.
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1.2
Setup of Main Hardware
Set the base of the NOVA View on the lab bench. Use of the placement screws on the base is optional and should only be done immediately prior to cover installation.
Figure 1-7 NOVA View base Place the microscope in the space outlined on the base. Verify that the coverslip correction ring on the 40x objective is set to 1.
Figure 1-8 Microscope placement on base
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Figure 1-9 Coverslip correction ring set to 1 The microscope is shipped with two transport locks, one securing the objective platform and the other the camera sensor.
Figure 1-10 Microscope transport locks To remove the transport lock from the objective platform, first uninstall the slide stage. Loosen and remove the four Allen screws from the stage and set the stage aside.
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Figure 1-11 Slide stage screws Use an Allen wrench to loosen the screw on the top of transport lock. Turn the nut of the lock clockwise until it reaches its lowest height. Then turn the clamping rod clockwise until it can be removed. The Allen wrench may be useful in loosening the clamping rod.
Figure 1-12 Removal of transport lock from objective platform To insert the mirror unit, loosen the Allen screw underneath the mirror unit platform on the right side of the microscope and remove the dummy cassette. Then slide the mirror unit into place, and retighten the Allen screw to secure it into position.
Figure 1-13 Loosen mirror unit platform screw Page | 15 NOVA View Installation and Calibration Manual Rev.0
Figure 1-14 Insert mirror unit
Figure 1-15 Securing the mirror unit Verify that the magnification selector knob is pushed in all the way to achieve a 1X magnification. Also verify that the shutter is open by pushing the lever under the word “Shutter” all the way towards the open circle. If the shutter is not completely open, the field of view will not be illuminated properly. Both the shutter lever and magnification selector knob are located on the right of the microscope below the objective stage.
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Figure 1-16 Open shutter and 1X magnification Reinstall the slide stage by positioning it above the objectives, ensure that it sits flat, and bolt it down with the four Allen screws.
Figure 1-17 Reinstall slide stage Unscrew and remove the security device from the left side of the microscope in preparation for the installation of the camera.
Figure 1-18 Remove security device Remove the plastic cap from the camera and screw the camera onto the camera adapter. Page | 17 NOVA View Installation and Calibration Manual Rev.0
Figure 1-19 Camera connected to camera adaptor Then install the other end of the camera adapter to the microscope by loosening the side screw with an Allen wrench, fitting the adapter into position with the proper camera orientation, and retightening the side screw to hold it in place.
Figure 1-20 Installation of camera and camera adaptor to microscope To adjust the camera orientation, loosen the side screw, rotate the camera adaptor until the camera is orientated correctly, and retighten. The camera should be orientated so that the two red indicator lights are on the upper left hand corner.
Figure 1-21 Correct camera orientation
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Set the microscope control unit on its position on the base.
Figure 1-22 Microscope control unit placement on base Remove the LED unit from the lower box and check the voltage and fuse on the back of the unit. If the system is being installed within the US, 120V power must be selected on the back of the LED unit and a 6A fuse installed.
Figure 1-23 LED for US use with 120V selected
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Systems installed outside the US must switch this to 240V and install a 3A fuse. The fuse is housed below the power connection on the back of the LED unit.
Figure 1-24 LED for use outside of the US with 240V selected Open the fuse drawer and verify or install the proper fuse depending on the country of installation. An alternate fuse may be supplied in the instrument tool kit or in the cable box. Once the proper fuse is installed, close the drawer. If the correct fuse is not included, please contact your instrument supplier.
Figure 1-25 LED fuse drawer
Place the LED unit in the space outlined on the base. Page | 20 NOVA View Installation and Calibration Manual Rev.0
Figure 1-26 LED placement on base Place the computer in the last marked position on the base.
Figure 1-27 Computer placement on base Set aside the collimator as its installation is covered in the following section.
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1.3
Installation of Instrument Cables
There are many cables required to connect and power the components of the NOVA View. If cables are missed or not plugged in properly, system errors will occur. It is therefore recommended to neatly organize the cables and test the system prior to system cover installation. Velcro cable ties are provided for optional use. For a photographic guide to the NOVA View cables, refer to appendix A. Remove the stage control cable from its compartment and plug the 25pin serial cable into the vertical serial port on the rear of the computer.
Figure 1-28 Stage control cable plugged into the back of the computer Wrap the slack of the cables around the front of the microscope. Connect stage control cable #1 to the socket below the stage and stage control cable #2 into the socket to the left of the stage. Screw both cables in tightly.
Figure 1-29 Stage control cables connected to stage
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Connect the three microscope control cables (1 small, 1 medium, and 1 large) to their respective plugs on the rear panel of the microscope control unit, labeled Main 1, Main 2, and Main 3.
Figure 1-30 Microscope control cables connected to microscope control unit Attach the opposite ends of the 3 cables to their respective plugs on the right side of the microscope.
Figure 1-31 Microscope control cables connected to microscope
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Locate the mirror unit cable. Plug one end into the fluorescence mirror unit, located on the left side of the microscope beneath the objectives. The plug faces the backside of the instrument.
Figure 1-32 Mirror unit cable plugged into fluorescence mirror unit Plug the other end of the mirror unit cable into the rear of the microscope control unit, in the upper right plug labeled “RFACA/FW1.”
Figure 1-33 Mirror unit cable plugged into microscope control unit
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Locate the RS232 cable with the USB adaptor. This cable connects the microscope control unit to the computer. Plug the USB adaptor end into an available USB port on the back of the computer.
Figure 1-34 RS232 cable with USB adaptor plugged into the back of the computer Connect the RS232 plug into the front panel of the microscope control unit.
Figure 1-35 RS232 cable connected to the microscope control unit
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Install the dongle into an available USB port on the back of the computer. The dongle is shipped inside the tool kit. The software will not initiate without the dongle.
Figure 1-36 Dongle installed in the back of the computer Locate the USB LED cable. Plug the USB LED cable into the back of the LED unit and plug the end with the USB into the back of the computer.
Figure 1-37 USB LED cable plugged into the back of the computer
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Install the handheld LED control unit by plugging it into the front of the LED unit.
Figure 1-38 Handheld LED control unit connected to the front of the LED unit Connect the camera to the system by plugging the fire wire cable into the camera. The upper port is preferred but either of the ports can be used. Avoid hot starting, plugging in the camera while the system is on, as this can cause the ports to malfunction.
Figure 1-39 Fire wire cable plugged into the upper port on the camera
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Plug the opposite end of the fire wire cable into the upper port on the back of the computer. Be careful to avoid accidentally plugging the fire wire cable into the Ethernet port as the incorrect connection will lead to a camera connection error at software initiation.
Figure 1-40 Fire wire cable plugged into the back of the computer To install the collimator, remove the red cap and insert the nozzle of the collimator carefully into the socket on the back of the microscope unit. Fix the collimator into position by tightening the two Allen wrench screws.
Figure 1-41 Collimator installation The fiber optic cables are housed under the computer keyboard in the shipping boxes. Locate the cables and remove the protective red caps. Be careful not to bend the optical cables against their natural curve.
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Loosen the knobs on the top of the collimator and insert the green marked optical cable into the rear port of the collimator with the corresponding green dot on the knob. Tighten the knob to hold the cable in place. The cable should be inserted just enough so that the silver lead is hidden.
Figure 1-42 Installation of green fiber optic cable into collimator Loosen the knobs on the front of the LED unit and insert the other end of the green marked optical cable into the port with a green dot on the knob. Insert the cable completely, until it hits the end stop, and then tighten the knob. Then insert the unmarked cable into the remaining port on the collimator just enough to hide the silver lead and tighten the holding knob. Insert the other end of the cable all the way into the left port on the front of the LED unit and tighten the knob to hold the cable in place.
Figure 1-43 Fiber optic cables plugged into the LED unit
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The collimator best fits inside the system cover when positioned with the fiber optic cables pointing downward. Loosen the two side screws on the rear of the nozzle of the collimator with the white handled Allen wrench, rotate the collimator into position, and retighten the screws.
Figure 1-44 Collimator position for system cover installation Connect the base to the monitor by inserting the base into the stand and tightening the screw on the bottom of the base.
Figure 1-45 Connecting the monitor base
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Plug in the power cable and monitor cable into the back of the monitor. Check that the monitor power switch on the back of the monitor, near the power cable, is in the on position.
Figure 1-46 Monitor with monitor and power cables plugged in and power on Plug the monitor cable into the back of the computer.
Figure 1-47 Monitor cable plugged into the back of the computer
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Plug the keyboard and mouse into the available USB ports, preferably on the back of the computer. If a USB to PS/2 port adaptor is available for the mouse, plug the mouse into the available port in the back of the computer.
Figure 1-48 Mouse with USB to PS/2 port adaptor
Figure 1-49 Mouse and keyboard plugged into the back of the computer Plug in power cords to the rear of the LED unit, microscope control unit, and computer. Connect any accessories included with the system (printer, handheld barcode scanner, USB hub, etc.). If the accessories require the use of USB ports, the cable configuration outlined in this manual may be modified accordingly.
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Check that the main switches on the LED, monitor, and microscope control unit are powered on. Plug the four power cords for these units and the computer into the power strip. A cord adapter may be needed depending on the cord configuration and country of installation.
Figure 1-50 Four system power cords plugged into the power strip If a backup power supply is provided, the power strip or four power cords should be plugged into the back of the power supply unit. Plug in and power on the backup power supply. To power on the system, switch on the power strip. This will turn on the components. Power on the computer and monitor to initiate Windows Vista. Check the handheld LED unit to verify that both wavelengths (400λ and 490λ) are displayed. Note that the percentages of use for each are not relevant at start up.
Figure 1-51 Handheld LED unit display at start up The monitor brightness should be set to at least 90% and display resolution must be set to the maximum (1280x1024) for the software to startup properly. Continue on to software initiation.
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Software Initiation
CH.2
CHAPTER 2: Software Initiation Initiate the software by double clicking on the NOVA View icon on the desktop.
Figure 2-1 NOVA View desktop icon The software can also be initiated by double clicking on the executable application file in the NOVA View software folder (Computer: New Volume (D:):NOVA View).
Figure 2-2 NOVA View executable application file
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Enter “service” as the user name, select the return button on the keyboard, and enter “2311” in the password field. A message will appear stating that service mode is enabled, select OK. If this message does not appear, service mode was not properly initiated and the software must be restarted with the service login and password.
Figure 2-3 Service mode initiation The software undergoes a self test at startup to check that all devices are connected and recognized. The stage will also home at this time. If a connection error occurs, power down the system and check the corresponding cables. Refer to the troubleshooting guide in Chapter 8 for further instructions. Service mode should only be used by service engineers as it allows for setting changes. Login for users and non service personnel only requires user identification information, no password is needed. Verify the current software version on the home screen. Contact INOVA technical support with questions regarding current software version. Refer to the Appendix D if NOVA View software is not installed or an upgrade is required.
Figure 2-4 Software version displayed on home screen Record software version and build numbers for the NOVA View-GUI software and each module. These can be found in the Version tab in the Settings window. To open the settings tab, select the settings button from the set up tab.
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Figure 2-5 Example version tab
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Centering the Optical Light Pathway
CH.3
CHAPTER 3: Centering the Optical Light Pathway Centering the light pathway ensures that light is evenly dispersed within any given image and is crucial for accurate image analysis of fluorescent intensity. This procedure requires one slide of any type. The slide masking will be used to center the optical light pass within the field of view. Locate the field iris diaphragm lever stored in the tool box. The lever regulates the location and diameter of the light pathway. To install, insert the lever into its position on the rear right hand side of the microscope and screw into place until tight. Push in the lever.
Figure 3-1 Installation of field iris diapraghm lever Place the slide in position one on the slide stage.
Figure 3-2 Slide loaded on stage in position one
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In the software, select the Image Mode tab and switch on Live image mode. Switch to the FITC channel and 40X magnification.
Figure 3-3 Live image mode in FITC with 40X magnification activated Using the navigation buttons, move to the masking of the slide. Click the green arrows to move the stage is the corresponding direction. Left click to move the stage in small steps, right click for larger steps.
Figure 3-4 Live image mode navigation buttons
Figure 3-5 Objective focused on slide masking
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To ensure correct scanning and analysis, the field iris diaphragm must be centered in the field of view. Reduce the diameter of the field iris diaphragm by pulling the lever out.
Figure 3-6 Lever pulled out With the lever pulled out, a small green octagon should appear on the monitor. Use the knob on the right of the microscope to bring the image into focus. The octagon should be centered in the field of view.
Figure 3-7 Focused and centered octagon
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If the octagon is not centered, use the white handled Allen wrench to adjust the two centering screws located above and below the lever. Make slight adjustments and observe the movement of the octagon on the monitor.
Figure 3-8 Adjusting the centering screws Once the field iris diaphragm is centered, push in the lever. The lever should remain pushed in for proper system use.
Figure 3-9 Lever pushed in
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With the lever pushed in, the field of view should be evenly illuminated on the screen.
Figure 3-10 Evenly illuminated field of view Deactivate Live image mode by selecting the Live Image button again. Always deactivate Live image mode before starting a new project or shutting down the system.
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System Cover Installation
CH.4
CHAPTER 4: System Cover Installation To install the system cover, exit the program. Shut down the computer and power off the system. Remove the four Allen screws from the stage. Loosen and unplug the two stage control cables. Wrap the cables around the front of the microscope and position the plugs near the objective stage so that they can be accessed once the cover is secured.
Figure 4-1 Stage removed and cables in an accessible position
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The handheld LED control unit, keyboard, and mouse must be accessible once the system cover is installed while the power cables and monitor cables require connections external to the system. To achieve this, there is a small opening in the back left corner of the base for cables to fit through. Position the three power cables (computer, LED, and microscope control unit), monitor cable, handheld LED control unit, keyboard and mouse cables towards the back left of the base and secure with a Velcro cable tie.
Figure 4-2 Power cables, monitor cable, handheld LED control unit, keyboard and mouse cables positioned towards the back left of the base Fix the rest of the cables so that they are all situated inside the base. Cable ties may be used to fix the cables into place. Be cautious of the ventilator fans inside the cover, located inside on the right and left sides. Position the cables low or away from these fans to prevent them from getting caught.
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Remove the Nova View system cover from the box. Connect the ventilator fans to the ventilator power cable. Attach the cables with corresponding labels (cables are marked with Xs and Os) and ensure that there is good contact between the leads.
Figure 4-3 Ventilator power cable attached to corresponding ventilator fan Plug in the power cord to test the fans. The ventilator fans should immediately power on.
Figure 4-4 System cover with both fans plugged in
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Once the fans are verified to be working, unplug the power cord and install the system cover on top of the base.
Figure 4-5 Installed system cover Check that the holes along the base align with the holes around the bottom of the cover. With the cover in place, the power cables (including the ventilator power cable), LED control unit, and monitor cable should be exiting through the opening in the back and the two stage control cables should be accessible.
Figure 4-6 Cables exiting through the back of the system cover
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To reinstall the slide stage, position the stage on top of the microscope lens stage so that it sits flat and that holes for the bolts align. It may be necessary to move the microscope slightly in order to properly position the slide stage. Connect the stage control cables and bolt down the stage.
Figure 4-7 NOVA View system with cover and stage installed Plug the ventilator power cord into the power strip and/or back up power supply. Fix the screws around the base of the cover as well as the smaller screws around the front panel of the computer (optional). Power on the system and computer. Next, complete the stage calibration.
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Stage Calibration
CH.5
CHAPTER 5: Stage Calibration Stage calibration consists of two parts, the X & Y calibration and the Z calibration. The stage moves in the X & Y direction to home the stage and image the slide wells during automatic scanning. The microscope objectives move up and down in the Z direction to achieve proper image focusing. Both calibration procedures are critical for optimal scanning and imaging by the NOVA View system.
5.1
Stage Calibration – X & Y
The stage uses coordinates to move in the X and Y directions to specific locations. During automatic scanning, the stage moves above the objectives to image precise regions of each well. This procedure uses a slide profile to check the alignment and measure the offset, if any. The homing of the stage is then adjusted so that each profile is aligned. This procedure requires an INOVA 12well or 6well HEp-2 slide. In the example, an INOVA 12well HEp-2 slide is used. Start the software in service mode. Enter “service” as the user name. Password is “2311.”
F i g u r e 5-1 Service mode initiation Load an INOVA 12well HEp-2 slide into position one and do not use the slide stage cover.
Figure 5-2 INOVA 12well HEp-2 slide loaded into position one
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In the Set Up tab, select New Project to open the slide selection window. Program a 12 well HEp-2 slide in the first position by selecting the slide name in the list under Slide 1. Enter a Project identifier, and click the green checkmark.
Figure 5-3 Slide selection window with 12well HEp-2 slide in first position Switch to the Image Mode tab and activate Live Image Mode. Select the 40X objective and enter a Z pos value of approximately 6000 to move the objective closer to the slide. The software will default to imaging Slide 1 Well 1 which is used in this example but, any well can be used can be used to complete this procedure.
Figure 5-4 Live image mode activated with 40X objective selected and focus height entered
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Visually check that the light is centered within the well. If adjustments are necessary, note the original X & Y coordinates from the well center. These can be found on the left hand control board next to X Pos and Y Pos.
Figure 5-5 Original X & Y coordinates Example: Original Coordinates X Pos = 13702 Y Pos = 9304
Using the green navigation arrows, move the stage so that the light is centered within the well. Left click to move the stage in small steps, right click for larger steps.
Figure 5-6 Live image mode navigation arrows
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View the well from directly above to visually center the light. Note the X & Y coordinates for the corrected position. Find the offset by subtracting the corrected coordinates from the original coordinates.
Figure 5-7 Corrected X & Y coordinates Example: Corrected Coordinates X Pos =14002 Y Pos = 9104 Offset X Pos = 13702 – 14002 = -300 Y Pos = 9304 – 9104 = 200 Deactivate Live Image mode and switch to the Set Up tab. Select the Settings button and the Devices tab.
Figure 5-8 Devices tab in Settings window
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Select stage settings and note the Zero coordinates in the Zero Point tab.
Figure 5-9 Zero point tab in stage settings window Add the offset values to these Zero coordinates. If a negative difference value was calculated, subtract it from the zero coordinates. Example: Corrected Zero Coordinates Zero X = 109458 + (-300) = 109158 Zero Y = 72393 + 200 = 72593 Input the corrected Zero Coordinates in the Zero point tab.
Figure 5-10 Corrected zero coordinates in zero point tab in stage settings window
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Select Apply and Save, then select the green checkmark to exit the settings. Exit and restart the software in service mode. The software must be restarted after any setting change prior to instrument use. To check the calibration, select New Scan, enter a 12-well HEp-2 slide and a Project identifier. Go to the Image Mode tab, activate the Live image mode and select 40X magnification. The stage should move so that the light source is in the center of well 1. Adjust the Z Pos value to 6000. If the light source is centered in the well, continue on to the Z calibration. If it is not properly centered, restart this procedure.
5.2
Stage Calibration – Z
The objectives move up and down in the Z direction, relative to the slide stage to achieve a focused image. During automatic scanning, the software relies upon a default Z value to capture clear and focused images. This procedure determines the default Z setting across a level stage. If the stage is not level, hardware adjustments will be necessary. To complete the Z calibration, two DAPI stained INOVA 12-well HEP-2 slides and the 1.5mm Allen wrench provided in the tool kit, are required. Load the slides into positions one and four. The slide stage cover is optional for this procedure.
Figure 5-11 Two DAPI stained INOVA 12-well HEP-2 slides loading into positions one and four
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In the software, select New Scan. Enter a 12-well HEp-2 slide in positions one and four and enter a Project identifier.
Figure 5-12 Slide selection window with 12-well HEP-2 slides entered in positions one and four Select the Image Mode tab, activate Live image mode, and use 40X magnification in DAPI for this procedure. From the drop down box, select slide 1, well 12.
Figure 5-13 Live image mode activated with 40X magnification and DAPI selected for viewing Slide 1 well 12
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The stage will move to center the light source beneath that corner well. Use the Z+ or Zbuttons or click on the image and use the mouse scroll bar to focus the image.
Figure 5-14 Live image mode Z+ and Z- buttons Once the nuclei are in crisp focus, note the Z-Pos value for that corner.
Figure 5-15 Focused Z-Pos value for Slide 1 well 12 From the drop down box select slide 1 well 7, then using the Z+ and Z- buttons or the mouse scroll bar, focus the image and note the Z-Pos value for this position.
Figure 5-16 Focused Z-Pos value for Slide 1 well 7 Page | 58 NOVA View Installation and Calibration Manual Rev.0
Follow this same procedure for slide 4 well 1 and slide 4 well 6. Now compare the focused Z value positions for all four corners. The stage is considered level if all four of these values are within 100 units of each other. With a level stage, calculate the average Z-Pos value. If the values are not within 100 units of each other level the stage before continuing with this procedure. Note that the 100 unit difference is an acceptable limit but does not guarantee proper automatic focusing. The tighter the four focus values are, the more level the stage is over the objectives. An unlevel stage can cause problems with focusing and increases runtime. Example: Slide 1, well 12 – 5914 Slide 1, well 7 – 5980
Slide 4, well 1 – 5960 Slide 4, well 6 – 5942
5980 – 5914 = 66 <100, within 100 units Average = 5949
Select Settings, Test, ANA, and input the calculated average as the Default focus setting. Rounding to a whole number is acceptable.
Figure 5-17 Entering the ANA default focus setting into the test tab in the settings window
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Also, select CALIBRATE and input the same calculated average for the default focus.
Figure 5-18 Entering the CALIBRATE default focus setting into the test tab in the settings window Press the green checkmark and restart the software. Automatically scan the four outer corners to check that the stage calibration is complete. Select New Project and enter a 12 INOVA well HEp-2 slide in position one and four and a Project identifier. Select the green checkmark. Select Sample Data and enter a SID and dilution in slide 1 well 7, slide 1 well 12, slide 4 well 1 and slide 4 well 6.
Figure 5-19 Sample Data window with a SID and dilution entered in slide 1 well 7, slide 1 well 12, slide 4 well 1 and slide 4 well 6
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Select OK. The Set Up tab will display the four outer corners with the entered sample data and is ready for scanning. Select Start Project Scan.
Figure 5-20 Set up tab with sample data entered in the four outer corners Watch the scanning to ensure that the software is easily able to capture focused images from all four corners. If all four corners were scanned by the software without difficulty or fault, the calibration is complete. To level the stage, navigate to a corner with an outlying Z value. Set the Z-Pos value close to the Z target, such as a value closer to the other three corners. Type the target value into the Z pos box and press enter
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Using the 1.5mm Allen wrench, make very small adjustments to the corner set screw until the DAPI stained nuclei are in focus.
Figure 5-21 Adjusting set screw with Allen wrench to focus the image Repeat with the other three wells. Minor adjustments will affect the focus values for all the other corners, so small adjustments are critical. While adjusting the corner set screws, check that the frame is sitting level by pushing on the opposite corners. If there is a physical unbalance between any of the corners, this will alter the Z height and impair the system’s ability to automatically focus. Once adjustments have been made to all four corners, start from slide 1 well 12 and note the Z-Pos value at which the image is actually in focus. Note the values for the other three corners as well and recalculate the average. The tighter these values are to an average, the better. If further manual adjustments are necessary, continue making small height adjustments with the Allen wrench until an average value at which all four corners are approximately in focus is achieved. This average Z-Pos value will be set as the default focus height. Select the Settings button and open the Set Up tab. Change the default focus for the ANA and CALIBRATE profiles to the average Z-Pos value obtained. Press the green checkmark and restart the software following the setting change. Automatically scan the four corners and verify that the software can capture images of the four wells without difficulty. Once this is confirmed, the stage calibration is complete.
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FITC Light Intensity Calibration
CH.6
CHAPTER 6: FITC Light Intensity Calibration The NOVA View measures light intensity in light intensity units (LIU). Calibration of the FITC light intensity standardizes the analytical sensitivity of the instrument relative to a master instrument, thereby minimizing instrument to instrument variation. This procedure measures the FITC output of the system in LIU, calculates a factor relative to the target value generated by the master instrument at manufacturing, and incorporates the factor into the software settings. The FITC light intensity should be calibrated at installation, during preventative maintenances, and at any major service or breakdown. Load one 6-well INOVA NOVA Fluor™ calibration slide (P/N: 508011.3) into position one. Cover the slide stage. Six data points, or wells, is the minimum required for this procedure. Software must be in service mode to complete this procedure. Restart software to enter service mode if necessary. Select New Project and in the slide selection screen, select INOVA 6w Calibration Slide in position one. Enter slide lot number and add “calibration” to the project name. Select the green checkmark to continue.
Figure 6-1 Slide selection window with one INOVA 6w Calibration slide entered Select Sample Data and select the Calibrator button for each well. Select OK.
Figure 6-2 Calibrator button
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Figure 6-3 Sample Data window programmed with six Calibrator wells Select Start Project Scan. Beads will come in and out of focus in grey scale and then focus in FITC to analyze an image and calculate the LIU for each well. The bottom toolbar displays the image analysis including focusing, bead count, and exposure timing.
Figure 6-4 Calibration well scanning in grey scale
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Figure 6-5 Calibration well scanning in FITC Once the scan is complete, select Intensity from right hand column in the Set up tab to display the LIU result for each well.
Figure 6-6 FITC intensity results displayed in the Set up tab Calculate the average light Intensity for the instrument by averaging the intensity results of at least six calibration wells. To calculate the FITC intensity correction factor (CF) divide the average light intensity by the target LIU. Check the certificate of analysis for the lot specific target LIU.
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Example: CF = (average system LIU)/(target LIU) Average system LIU = 1559 CF= 1559/1350 = 1.155
Target LIU=1350
To input the FITC intensity CF into the settings, select the Settings button and open the Devices tab. Enter the CF into the FITC Intensity field, select the green checkmark, and restart the software.
Figure 6-7 FITC intensity CF in the Devices tab Technician can now complete a test run or begin training.
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Backup of Important Files
CH.7
CHAPTER 7: Backup of Important Files Certain files should be backed up during installation and preventative maintenances as they can be useful in the case of an instrument failure. A copy should be saved on an external drive at the customer site as well as with the service engineer.
7.1
Settings File
A notepad file of the instrument settings is automatically created and saved in the NOVA View software folder. As this file overwrites itself each time there is a change to the settings in the software, maintaining a copy of the proper settings is crucial. Note that the only way to change the settings in the software requires the use of Service mode. Changes made to the notepad settings file will not be reflected in the software. To access the settings file, open Computer, New Volume (D:), NOVA View and scroll down to file named NOVA View and labeled as SETTINGS File.
Figure 7-1 SETTINGS File located in the NOVA View software folder Save a copy of this file to an external drive or USB drive that will remain at the customer site. The service engineer should also retain a copy for his or her records.
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7.2
PRO File
The PRO File writes the instrument serial number to the dongle and allows access to the software. Without the dongle and this file, the software will not initiate. To access the PRO File, open Computer, New Volume (D:), NOVA View and scroll down to file named NOVA View and labeled as PRO File.
Figure 7-2 PRO File located in the NOVA View software folder Save a copy of this file to an external drive or USB drive that will remain at the customer site. The service engineer should also retain a copy for his or her records.
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Performance Characteristics and Specifications
CH.8
CHAPTER 8: Performance Characteristics and Specifications Dimensions (not including monitor, keyboard, or accessories) 64cm W x 64cm L x 23cm H Weight 145lbs (65kg) Operating Conditions Instrument operates at room temperature (18-25°C). Supply Voltage 120 to 240V, 50/60 Hz, 6A (US) or 3.5A (EU) Performance Specifications Scans and archives images from single well in one minute. Capacity The NOVA View instrument can automatically scan up to four processed IFA slides per session. Quality Control Control materials should be included with each instrument scan. Reagents The staining of slides with DAPI (diamidino-2-phenylindole) is required for use on the NOVA View instrument. DAPI is a blue fluorescent probe that selectively binds to double stranded DNA and allows staining of the nuclei with little background from the cytoplasm. The instrument uses the DAPI fluorescence to focus on the nuclei of the cells during scanning, prior to the FITC analysis. Interface Capabilities System is interfaced with QUANTA Link™.
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Troubleshooting
CH.9
CHAPTER 9: Troubleshooting Start Up Issue:
Check/Correct:
Notes
Stage does not home properly during software initiation. Microscope connection error at software initiation.
Stage control cable is not plugged in correctly. Check serial computer connection and both stage plugs. Check the microscope control cable connections (Main 1, 2, and 3) at both the Microscope control unit and the Microscope. Check RS232 cable with USB adapter at both the microscope control unit and computer connections. Check that the microscope control unit is powered on. Check the USB LED cable. Check that the LED unit is powered on. Check handheld LED unit display. Check that the LED is connected in the Device Manager (pEx USB COM Port) and that there are no port conflicts. Check firewire cable. Both red lights on the camera face must be illuminated. Check that the camera is connected in the Device Manager. Device Manager must register the camera as a Kappa Imaging Device (Kappa DX4-285FW). If the Device Manager lists “Generic camera,” the camera driver needs to be re-installed. Security dongle is not detected. Check that it is properly plugged into a computer USB port. Open the NOVA View software folder and verify that the PRO file is present. If it is missing, restore it with a back up copy. If no backup copy is available, contact INOVA technical support.
Stage Cable 1 powers the X direction of the stage. Cable 2 powers the Y direction. The blue power indicator light on the USB adapter connected to the RS232 cable should be illuminated if properly plugged into the back of the computer.
LED connection error at software initiation.
Camera connection error at software initiation.
“Copy protection not found, exiting program” error.
Error occurs when user exits and immediately reinitiates the software. Error can be avoided by waiting a short period of time before reinitiating the software.
The component drivers are provided on the Service CD.
Dongle is shipped in the tool kit.
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Scanning Issue: “No DAPI-FITC filter set found, scan complete without reading” error.
No FITC or DAPI images displayed during automatic scanning. Image screen remains black and no cells are imaged. Problems focusing during automatic scanning.
Field of view not illuminated properly.
Result Issue: High incidence of result misinterpretation.
Check/Correct:
Notes
Check mirror unit cable and restart software. Check that a DAPI-FITC filter is programmed in the microscope settings. Check the fiber optic cables. Check the handheld LED unit for functionality of the LED unit. The 400nm and 490nm wavelengths must be registered by the unit. Clean excess mounting media off the slide, especially the bottom. Check the Z default value in Live image mode. Clean the 40x objective and check that it is in the proper position. Check that 40x objective correction ring is set to 1. Check that the slides are seated properly. Check X & Y calibration. Check that magnification selector knob is pushed in to achieve 1x magnification. Check that the fiber optic cables are installed properly. Check that the light source is not imaging part of the slide masking. Check that the field iris diaphragm lever is centered and pushed in during imaging. Check that the shutter is completely open.
Check/Correct: Check result images for proper illumination and DAPI/FITC staining. Check that the field of view is illuminated properly. Confirm images from automatic scanning in Live-Image mode.
If mirror unit cable is properly plugged in, DAPI and FITC buttons will be available when Live-Image mode is activated. When experiencing problems with automatic scanning, confirm that Live-Image mode is working properly. If the LED is not registering the two wavelengths, power down the entire system and restart. The first grayscale image during automatic scanning is the instrument attempting to focus on the cells. This process can take significantly longer and affect interpretation if there is excess mounting medium or if the Z default value is set incorrectly.
Notes Positive samples should have 5 images taken per well.
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Instrument Cables
APX. A
APPENDIX A: Instrument Cables
Some instruments may have USB LED cables that instead look like this:
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Checklist of Components Shipped with System
APX. B
APPENDIX B: Checklist of Components Shipped with System Hardware
Quantity
Microscope Unit IX81
1
Slide Stage
1
Microscope Control Unit IX2–UCB-2
1
CoolLED PreciseExcite Kappa Digital Camera
1 1
Collimator
1
Monitor + Base
1
Fiber Optic Cable
2
Shuttle PC
1
Mouse
1
Keyboard
1
Stage Control Cable
1
RS232 Cable with USB Adapter
1
Firewire Cable
1
USB LED Cable
1
Microscope Control Cable
3
Mirror Unit Cable
1
Monitor Cable
1
Dongle
1
Handheld LED Control Unit
1
Power Cord
4
Took Kit with Allen Wrench
1
Power Strip
1
System Cover
1
Base
1
Ventilator Fan
2
Ventilator Power Cord
1
Panel Screws
8
System Cover Screws
20
Software Installation CD
1
Service CD
1
Slide Stage Cover (if not attached to microscope)
1
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Installation Record
APX. C
Customer: _______________________
APPENDIX C: Installation Record
Address:
NOVA View
_______________________ _______________________ _______________________
Phone: Installation Record Serial #: GUI Version: GUI Build: ANA Version: ANA Build: Check boxes for damage in transit. Unpack components and inspect for visible damage.
Place the base on the lab bench. Position the microscope on the base. Set the cover glass correction ring to 1. Remove security devices from microscope. Install mirror unit. Verify that 1X magnification is selected and that the shutter is open. Install slide stage, connect stage control cables, and screw stage into place. Install the camera adapter and camera with the correct orientations. Set the LED unit on the base. Verify the power and fuse. USA: 120V, 6A. EU: 240V, 3A. Set the microscope control unit and computer on the base. Attach stage control serial cable to the computer. Connect microscope control cables and mirror unit cable. Connect the microscope control unit and computer with the RS232 cable/USB adaptor. Install dongle in a USB port on the back of the computer. Connect LED unit to the computer with the USB LED/USB extension cable. Install handheld LED control unit. Connect power cords to the LED unit, microscope control unit, and computer. Connect firewire cable to camera and computer ports. Install the collimator and fiber optic cables. Reposition collimator as necessary. Attach the monitor base to the monitor. Connect monitor cable and power cord. Plug in mouse and keyboard to the appropriate computer ports. Connect instrument accessories (optional). Plug the four power cords for the computer, LED, microscope control unit and monitor into the backup power supply or power strip. Plug in and power on the backup power supply unit or power strip. Power on the system and initialize the software. Verify software version. Complete the field iris diaphragm lever installation and center the optical light pathway. Install the System Cover. Complete the stage calibration (X & Y and Z). Complete FITC Light Intensity calibration. Record the correction factor. Correction factor: Record instrument version/build numbers. Backup the SETTINGS File and the PRO File. Train user(s) in the operation and routine maintenance of the system. Installation completed by:
Date of completion:
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Instructions for Software Installation and Upgrade
APX. D
APPENDIX D: Instructions for Software Installation and Upgrade NOVA Views are supplied by INOVA with the current version of software. Software upgrades will be announced and distributed by INOVA through technical bulletins and International Marketing Updates (IMUs). Technical bulletins will provide specific instructions for each software upgrade and may include setting changes. It is the responsibility of the service provider to upgrade the systems they support. Below are general instructions for software installation and upgrades. First, delete the current NOVA View desktop shortcut to avoid confusion between shortcuts as the patch installation will create a new shortcut. It is also recommended to save a copy of the SETTINGS File (see section 7.1) as the patch may overwrite instrument specific settings, such as the default focus. Acquire the installation executable file (generally named: NOVA View-version numberInstaller) from a disc or flash drive. Insert the disc or plug in flash drive to the NOVA View computer and access the file.
Double click on the installer in the NOVA View software folder to begin the software upgrade. An installation wizard will open.
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Select Next to continue.
. Select Browse to chose the destination folder where the previous version is located.
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Select OK and then Next to continue.
Select Next to continue.
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Select Next to continue.
Select Install.
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The instrument specific default focus setting for the ANA method from the previous software version is automatically copied into the ANA method for the updated software version. This is the only method automatically updated so other methods (ie: CALIBRATE) will need to be updated manually. Select OK to continue.
The update is complete. Select Finish to exit the installation application. Check that the patch has been properly installed by initiating the NOVA View software in service mode.
Initiate the software in the service mode by entering “service” as the Login and “2311” as the password. Select the green checkmark to continue. In the Set Up tab, verify the correct software version number on the right hand side.
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Select the Settings button and open the Version tab to verify the settings and build numbers according to the specific upgrade . Change the default focus (z) values for the CALIBRATE test to match the focus height set in the ANA tes. Select the green checkmark to exit the settings. Exit the software and restart in user mode to operate the NOVA View. A copy of the updated SETTINGS File (see Section 7.1) should be saved to an external drive or USB drive that will remain at the customer site. The service engineer should also retain a copy for their records.
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INOVA Diagnostics, Inc. 9900 Old Grove Road San Diego, CA 92131 United States of America Technical Service (U.S. & Canada Only) : 877-829-4745 Technical Service (Outside the U.S.): 00+1 858-805-7950
[email protected] www.inovadx.com Page | 85 NOVA View Installation and Calibration Manual Rev.0
