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Software Manual
10.9.6
Chapter 10 – Intensity Analyses
Calibration
The first step is to calibrate the contribution of the fluorochromes to the different color channels.
Depending on the number of different fluorochromes to be analyzed, different reference samples
stained with only one fluorochrome have to be prepared.
Calibration
1. Acquire multi-color reference images of the reference samples under the same imaging
conditions as used later for the experiments with your multi-labeled samples.
It is possible to use multi-labeled specimen for reference purposes in case monolabeled structures are clearly resolved in the images.
For example, the following filters have to be used to unmix CFP and YFP:
— CFP exciter (for one color channel)
— YFP exciter (for the other color channel)
— dual-band dichroic mirror
— dual-band emitter
2. Load the acquired reference images into the image buffer and click on the Calibration
button. This opens the Measure Ratio (Unmixing) dialog window.
3. Press Define ROIs to open the respective dialog box in order to define two ROIs. The
first one has to be within an area where the fluorochrome is visible while the second ROI
is used for background correction. The ROIs are set in the same way as in the Image
Define ROIs... menu (Chapter 10.4, Regions of Interest – ROIs).
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