Instructions for Howto Scan µarrays

Transcription

1 Instructions for Howto Scan µarrays Introduction After probing the µarray slides with samples, one is now ready to scan them. To scan a µarrays slide is too convert the biological information trapped on the slide into digital information for data analysis. We use a GenePix 4000B scanner ( or The act of scanning involves using a laser to excite the fluorescent markers on either the antigens or secondary antibodies and detecting the intensity of the fluorescence given off by these markers after being excited. The fluorescent intensity given off by the markers is dependent on three things, 1) the number of markers present, 2) the power of the excitation laser, and 3) the setting on the detector (PMT * ). For any given experiment, the entire set of µarrays slides probed in the experiment is scanned at the same laser power and PMT settings. Therefore, for any given scan session, the fluorescent intensity depends only on the number of markers present on the µarrays slides. Thus, in theory, we could quantify the number of antibodies from a sample that bind to a particular antigen. In practice, we use the fluorescent intensity for relative measurements, meaning, we report the results of our data analysis as sample A has autoantibodies with more/less/equal reactivity against these antigens when compared with sample B. Nonetheless, the data is quantitative and the fact that we could report absolute values makes the technique a very powerful tool. It just doesn t seem necessary at this point because our view of biology is not so precise as to require absolute values. Scanning a µarray Slide Launch GenePix 5.0 A program will search for the scanner and a screen might appear like the one pictured below. Uncheck the update reminder and click Continue. * PMT is an acronym for photo multiplier tube. It is a device that converts how much light striking the detector into a numeric value.

2 I ve added some tags (shown in red with their keyboard shortcut in parentheses) for the more common buttons or features used in the scan. Manual Brightness/Contrast Data Scan (Alt+D) Auto Scale Brightness/Contrast (Alt+T) Zoom Mode (Z) Hand Mode (H) View Scan Area (Ctrl+V) File Hardware Settings (Alt+Q)

3 Setting up the Scan Click Hardware Settings (Alt+Q) Adjust the Power for both channels (635 & 532) to 100%. Adjust the PMT settings for each wavelength to a value that maximizes the brightness of the signal intensity without saturating the detector. The spots will appear white when the detector is saturated. An optimal setting is where the entire µarray shows only a couple of saturated spots. The PMT settings represent the trade-off between signal and noise. One wants to have the Gain as high as possible where the spots are bright and not saturated. One also wants to keep the background level low. Unfortunately, the Gain setting enhances both the signal and the noise, thus, the trade-off. Set the Pixel Size to 10 µm Lines to average 1 (we only need to scan the slide once) Focus Position 0 µm The Scan area: (XXXX x XXXX pixels) and Scan duration: (XX seconds) are set by the scan area and the number of lines to average. Select Hand Mode (H). Select View Scan Area (Ctrl+V) and adjust the rectangle to the best guess for including all the spots on the µarray. It s best to make the border slightly larger so there is some error room because each slide will have the grid of spots in a slightly different location. One saves time by not having to adjust the frame for each slide. The savings may seem like a small amount, say 10 seconds, but over the course of an entire scan of 60 slides it adds an additional 10 minutes of time sitting in front of the scanner. The power setting adjusts how much power the laser will use to excite the sample. There are two collection wavelengths (635 nm and 532 nm) corresponding to red and green colors respectively.

4 Select File, Save Settings As (Alt+H). Name the settings for this scan session. It s a good idea to include the experiment name/number/date and the print date for the µarray slides. For example, here s the format I use: ScanSettings_expXX_YYYY_MM_DD_print_YYYY_MM_DD Note, since we re using the same settings for all the slides from a given experiment, it s important to save the settings in case one needs to come back and scan the same slides at a later date. Scanning the Slide Open up the scanner, lift up the slide clamp, and place the slide, label side up, into the scanning slot. Close the slide clamp. Close the scanner. The scanner will make some noise as it loads the slide into position to scan. Select Scan (Alt+D). The scanner will make some noise as the laser scans the slide. Select Zoom Mode (Z). One can zoom into the square region for a closer view of the µarray spots. After the scanner is finished, determine whether the settings are optimal for the experiment. Non-optimal settings would be incorrect PMT settings (spots are too dim or too many saturated spots) or the square is in the wrong place. Adjust the settings so they are optimal and save the settings. If the settings were not optimal on a scan, then one needs to scan the slide again. In this case, it s better not to save the image. If the scan settings are optimal, then save the settings (see above) one more time. Again, remember to keep the same scan settings for the entire set of slides.

5 Saving the Image Select File, Save Images (Ctrl+S). Choose single image tiff and check both channels (532 & 635) to have an image of the red and green intensities for each slide. We use the information from both channels for some analyses so it s best to collect it now if you re not sure. That way, you don t need to come back and scan all the slides again. Name the slide. It s a good idea to include the slide number and print date for the µarray slide. For example, here s the format I use: slxx_print_yyyy_mm_dd Continue Scanning the rest of the slides in the experiment.