Analyzing Slides
Version 2
Analyzing Slides:
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Open Scan Array Express (desktop icon)
If previous files are loaded in scan array, first close them by selecting “File” then “Close
All”
In scan array software, under “Configure & File”, select “File”
o Select “Open File”:
 Array Work/Fluor Scan Images
 Choose folder date
 Choose slide image
Select “Palette” in the bottom right hand corner
o Choose rainbow color
o Select “Ok”
Adjust the “Black threshold” and “Full color threshold” as appropriate (bottom right)
o Set the black threshold as high as possible without causing undue background
o Set the color threshold so the colors are distributed over the widest distribution
Select “Quantitate” (upper left)
o Click button “From .Gal File”
o Open appropriate Lectin GAL file:
 For 12 subgrid analysis: select “Lectin CDS 2v3.5g
Select “Adjust Template and Register Images” (lower left corner)
 First click on the center of the upper left subgrid
 Select “Zoom In” (upper right) and move in until you can see the spots
 Click and hold on the template grid and drag the template until the upper
left circle is perfectly over the upper left spot
 Rough adjust the size of the template grid by dragging the right edge and
also adjust the bottom template border so that the spots reside within the
white grid
 In the upper left scan array “adjust template” box select “rotates all
subarrays”
 Click on the lower, right subgrid
 Click and hold the template grid and drag/rotate until the columns are
lined up (the spots will not necessarily be be lined up yet)
 In the upper left scan array box select “Moves or resizes all subarrays”
 Click on the upper left subgrid, check the alignment if still good proceed
to next subgrid by clicking on the center of it, otherwise adjust the right
and bottom border again to get the template perfectly over the spots
 In the upper left scan array box select “Moves a subarray”
 Adjust each individual subgrid so that each is best fit over it’s appropriate
spots-it doesn’t need to be perfectly centered yet, just grossly correct or all
the data will be off.
Analyzing Slides
Version 2
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Select “OK” (bottom left) when done
 Note-once the grids have been adjusted in size and position
they stay grossly correct for future analyses as long as a
new gal file is not opened- so don’t import the gal file again
In the “Quantitate” window:
o Under “Auto find Spots” uncheck “Align Subarrays” and “Align Spots”
o Select “Adaptive circle” under “Quantitation method”
o Select “Total” under “Normalization method”
o Click “Align spots now
o Select “Adjust Template and Register Images” (lower left corner) and verify that
the template circles where placed around the visible spots accurately
o Hi OK
o Select “Start”
o At the top of the window select the “Cyanine5” tab
o Click on the upper left subgrid and zoom in until you can see the almost the entire
subgrid in the screen
o Adjust any template circle that needs it in position (click center and drag to
position) or size (drag edge to make bigger or smaller)
 Circles should be adjusted in a way that fluorescent artifacts have minimal
impact
 Affects of a particular circle adjustment can be seen by, selecting the
circle (click on it-cross hair appears), then clicking the spreadsheet tab at
the top of the window, a blue highlight indicates the data for that circle.
Look at the value for the circled spot- go back and highlight it’s replicate
and compare it.
 Note-once the project is saved you cannot record further changes in spot
position or fluorescent value so don’t use the save until you’ve finish
adjusting all 12 subgrid templates.
 Note2-don’t spend inordinate amounts of time in this process (bad
backgrounds or excessive pickiness will result in you spending huge
amounts of time on this process without changing the results -experience
will eventually guide you with selection of what needs attention and what
doesn’t and what changes things and what doesn’t. You can expect a
15secs per grid with the best images and 1-2 minutes per subgrid for even
really good images. Bad background or low image intensity due to sample
quality can create images that take large amounts of time. This is where
the replicates help you save time.
 Note3-sometimes the circle borders may looks like they don’t really ring
the circumference of the spot under low zoom power but if you zoom in
you’ll see that it actually does. Also, if the circle is consistently being
placed in on the replicates and they aren’t perfect by your criteria, then it’s
probably not worth changing them to be exactly perfect because the cost in
time versus the data payoff. You’ll see.
Analyzing Slides
Version 2
Save project as CSV file:
 Click on the “Spreadsheet” tab (top)
 Under “Configure & File” (left side)
o Select “File”
o Select “Save As”
 Select or make folder with appropriate scan date (the folder should be
stored in a folder called CSV results)
 Name according to distinguishable information; Date_comboID_slide #
o Example; 8-3-11_H201008_s1.CSV
(slide number helps with making consecutive comparisons)
o Select “Save”
Save project as Excel file:
 In the “ScanArray” window
o Click “View as Excel Spreadsheet” (bottom right)
 In the “Excel” window
o Select and highlight a blank row #7 and insert another blank row
 This puts the begin data marker at line 75 which is very important
for glycoanalysis
o Highlight the the entire document by clicking on the corner cell
o Select “File”
o Select “Save As”
 Select or make folder with appropriate scan date (the folder should be
stored in a folder called “Excel results)
 File name according to distinguishable information
o Date_comboID_slide #
o Example; 8-3-11_H201008_s1.xls
 Select “Save as type”
 Select “Microsoft Excel 97-Excel 2003”
 Select “Save”
 Close Microsoft Excel