Olympus IX70 SOP
Created: Virginia Vance, Apr 2013
General Set-up
1. Turn on monitor. Open CellSens Dimension from desktop icon.
a. Select “live” from camera control
b. Sensitivity “ISO 200”
c. Exposure time at lowest setting
2. Turn on Prior Optiscan II behind monitor
3. Turn on Olyumpus fluorescence lamp
4. Ensure that the lowest objective (X4) is set
5. Mount prepared dry sample facing down; mount wet slide facing up
6. Turn the dial on the filter cube turret underneath the objectives until “B” clicks into
place
7. Turn on OlympusTH-100; adjust light intensity to appropriate level
8. Use Prior motorized focus drive to bring the sample into focus
Fluorescent Imaging
1. Using appropriate excitation/emission spectra for the material construct or use a filter cube
capable of measuring its fluorescence.
2. Follow general set-up procedures. Disengage OlympusTH-100.
3. Turn dial on filter cube turret until the correct filter is in place.
4. In CellSens Dimension:
a. Sensitivity “ISO 1600”
b. Increase exposure time until the sample is visible. It may be necessary to turn off
ambient lighting
Quantitative
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Optimize fluorescent imaging conditions
Select an area of the sample. Use “Snap” to take an image
Repeat this over 10-20 different areas of the sample.
Save images as .vsi or .tiff
Open images using ImageJ software.
Select “File”>”Import”>”Image Sequence”>Olympus images
Select the number of images
For image “1/x” elect “Analyze”>”Histogram”>”No”>”List”>“Ctrl-A”>”Ctrl-C”>”Ctrl-V”
into Excel spreadsheet
Scroll right to view next image; repeat step 8 on a new page.
Take Column B of each page and transpose to a new page
Average each row
Transcribe this data into Origin software; generate a histogram