Scanner, Array
GENEPIX AXON SCANNER 4000B (B446)
Rules and Guidelines
Before use:
1. New user MUST be trained by the captain or present users
2. Log user information. To sign up to use this instrument, please go to the calendar at www.yahoo.com.
The user mail id is “axonscanner2”. The password is “botstein2”.
Instructions for use:
If the program is not open, select the shortcut titled “GenePix Pro 3.0” on the desktop.
Do not select the shortcut titled “GenePix Pro 5.0 because there is no USB key for the proper operation of this software.
The scanner should respond to your command within 2 minutes, indicating proper communication between scanner and computer with a flashing blue light on the top of the scanner.
Gently slide open the door of the scanner. The slide carrier will move automatically to the loading position. Once the carrier stops moving, the yellow eject light on top of the scanner will turn on,
indicating that it is safe to load your slide. Open the top half of the slide carrier by lifting the knobbed latch.
Place your slide into the slide carrier so that the chemistry side faces down. The optics of both the GenePix 4000 A & B are designed such that laser light is directed up from the bottom of the scanner.
Note that there are three star-sapphires on which the slide will rest. There is absolutely no need to push down on the slide. To ensure that the slide is resting flat against the sapphires, nudge the
semicircular pin to the left of your slide.
After closing the sliding door, the scanner is ready to scan your array. In the right column of icons, select the Hardware Settings dialogue box, depicted by a blue scanner with a yellow wrench. Also, in the
upper left corner, select Preview Scan
Typically, you will first select the low resolution Preview Scan button (the double triangle dialogue box located at the top of the right column) to acquire rapidly a rough representation (40um per pixel) of
the layout of the microarray.
After completing a preview scan, define the boundaries of the array by selecting the Scan Area dialogue box in the left column of icons (or you can select Ctrl+V). Adjust the boundaries of the box to fit your
array. "Save".
Now, you will re-scan your microarray at high resolution (10um per pixel). During this initial scan, you will optimize the hardware settings. Specifically, the hardware settings dialogue box controls the
amplification or gain of the photomultiplier tubes (PMTs), which is adjusted to maximize the dynamic range of the scanner. To change the amplification, increase or decrease the voltage of the PMT by typing in
a new voltage or by using the spinners to increase or decrease the current voltage.
For optimal PMT, set each PMT such that there are only a few pixels that are saturating in each image generated ie 1/1,000. Saturating pixels represent a condition whereby there are more photons detected
than the PMT can process or the output of the PMTs exceeds the range of the analog to digital converters. These pixels are visible as white pixels in the image; alternatively, you can determine the number of
saturated pixels from the histogram.
Do not dally in the optimization of PMTs. Energy from the laser will photo-bleach Cy5 more than Cy3, a small amount with every pass. When optimizing, do not abrogate the optimization run mid-course. This will
introduce Cy3 to Cy5 bias.
Another tool that you can use in optimization is the histogram. The histogram is found in the Histogram tab. It plots the distribution of all of the pixels from each wavelength for the currently displayed
images. Saturated pixels appear on the far right of the histogram, as saturation corresponds to an intensity value of 65535. It is important to realize that the histogram only plots the data for the currently
displayed image. If you have zoomed in on an image, the histogram represents only the region that is currently visible. The histogram is a useful tool to quickly see how much of the dynamic range is being
utilized. In addition, the histogram allows you to quickly see how much overlap you have in the red and green channels. This is an indication of color balancing. For example, the ideal scan is one whereby the
peak of the green histogram overlaps quite closely with the peak of the red histogram. This indicates that the two channels are quite similar in intensity. In reality, often one channel is slightly shifted
relative to the other due to it having higher background fluorescence. In practice, try to achieve a ratio as close to 1.0 as possible in the histogram while also visually confirming that the array image is
balanced, not too much green or red.
Scan your image. Do not change the PMTs during this final data scan. Once your microarray has been scanned, the next step is to save the image. Select the Save Images dialogue box (or you can select Ctrl+S)
in the right hand column. This will offer you a number of flexible options to control how your data is saved.
Save your image as a single-image TIFF file into a folder with your name on it located in the User’s File. Make certain that you select both wavelength channels 635nm and 532nm at the bottom of the popup
window.
Transfer your data as soon as possible. TIFF files are deleted every Monday at 5pm.
Troubleshooting
If the computer can not find the axonscanner, close the GenePix program and try again three times total. If on the third try the computer still can’t find the scanner, completely shut down the computer and
the axon scanner. Wait one minute, then turn on the axonscanner and then turn on the computer in that order. Next, select the GenePix Pro 3.0 shortcut. Look for the flashing blue light indicating a healthy
marriage. If no communication, repeat this process three times.
If on the third try there is no communication, insure that the pins of the SCSI cable are not bent and that the SCSI cable secured to both the computer and scanner. Initiate your scan.
If this does not work, check that the SCSI ID 4 does not conflict with any other SCSI devices.
If the spots in your microarray image demonstrate cusps of red and green in opposition to one another, this is an alignment problem. The first thing to do is to save the image should we need to call Molecular
Devices tech support. Select the Align Non-Axon Images dialogue box in the top left column or press F7 to realign the images.
If there are horizontal bands of green-only signal or red-only signal across the array, this is a power supply problem. The first thing to do is to save the image should we need to call Molecular Devices tech
support. Abort your scan and begin again. If the problem continues, abort your scan and shut off the computer, scanner and power supply. Turn on the power supply, scanner and computer in the order mentioned.
Open GenePix Pro 3.0 and initial your scan. If this does not work, there is a new power supply under Rob’s bench to replace this old power supply.
Manufacturer
Molecular Devices
For Service
Contact: Leslie Sun
Voicemail: 800.635.5577 x6054
Email: Leslie.Sun@moldev.com
or
Darren Marshall
Voicemail:408.431.5344
