Scanning Slides using Scan Array Express (Packard

By Stephen Riley,2014-05-29 12:39
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Scanning Slides using Scan Array Express (Packard

    Scanning_Slides, June 2005 NM

    Scanning Slides using Scan Array Express (Packard Bioscience

    BioChip Technologies Model ASCEX00)

A. Preliminary scanning

     Open Scan Array Express programme

    o Turn on Lasers and let warm for 15 minutes (Figure 1)

    o Laser 1- 633 nm (red), Laser 3- 543nm (green)

     Under Configure ; File

    o Select Configure (Figure 2)

    o Select Scan Protocols (Under Scanning)

    o Select FOAR03- Cy dyes (Arabidopsis full genome array (28K))

    (Figure 2)

    o Select #6 Tools

    Gently ease slide into chamber slot

     Run a Quickscan (Figure 3)

    o Press Start

Notes on scanning:

    Greater effects are made by adjustment to the Photomultiplier intensity (PMT) value than to the Laser Power (LP). Only one parameter should be adjusted, while the other is kept constant. As the LP (energy required to excite the fluorophore) reaches maximum excitation at values of 85 / 90%, LP is generally left constant at 90%.

During Quickscan, adjust the PMT dynamically.

     Small adjustments to the PMT make significant effects. To start, adjust

    setting 1 percent at a time. Note that increasing the PMT value increases the

    background and the spot intensities almost linearly.

     Try to maintain background values of not more than 200-220 all while

    saturating some pixels/spots. The slide generally appears more saturated than

    it really is after analysis. A spot/pixel is considered saturated at ~65,500

     It is better to lose a couple of spots due to saturation than to lose half the grid

    from being too light to detect.

    Black threshold adjustment on the screen does not affect the data to be saved

    When PMT levels have been optimized for the initial slide, scan each slide in the run individually. Generally, there is no need to recalibrate every slide in a single experiment. In fact, usually the same PMT/LP settings for all slides in a given experiment are adequate. Don’t forget to re-scan the slide used to optimize!

    Scanning_Slides, June 2005 NM

B. Final scanning

     Under Run

    o Select Scan

     Under Scan type

    o Select Run a scan protocol (Figure 4)

     Under Scan Protocol

    o Select FOAR03- Cy dyes

    o (Select “Image autosave protocol” as “default”)

     Click on Start

    During scanning note spot morphology, saturation levels, background levels, hybridization / local effects, etc…

    Re-do the quick scan and re-adjust the PMT/LP values if necessary but this should be avoided due to fluorophore quenching.

As mentioned, once the quick scan parameters have been set for one slide in a like-series,

    PMT/LP levels are simply left as they are for all the slides that follow.

C. Saving the scanned files

    Save the data output for each dye’s reading separately by first selecting the correct tab

    (Cy3 or Cy5).

     Under Configure ; File

    o Select File

    o Select Save as

    o Select a folder ex. My Documents/Arabidopsis/

     Name the file as follows “(slide barcode)_3.tif” or “(slide barcode)_5.tif

     Transfer data onto a CD for transfer to another computer and further processing

Scanning_Slides, June 2005 NM

Scanning_Slides, June 2005 NM

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