To generate and test a calibration table:
Mount the slide and 'blind focus' on the fluorophore. To 'blind focus,' close the field diaphragm and adjust the Z focus until the edge of the aperture is clear. Then open the field diaphragm to the point where you intend to have it for imaging the specimen.
Focus on the inside of the slide. (First focus on the slide surface, then focus about 10 micrometers within the slide.) Defocusing helps to minimize the effects of inhomogeneity on the surface of the calibration slide.
Ensure that all microscope and Resolve3D parameters are correct.
Collect an image and assess signal intensity. Determine an exposure time that will give a maximum intensity of about 3800 counts.
From the Resolve3D menu, choose Calibration | Make. The Calibration window is displayed.
Click the Calibration button at the bottom of the window.
After the test measurements are completed, histograms of CCD dark current, best-fit slopes, best-fit offsets, and correlation coefficients are displayed.
Adjust the histogram threshold bars to exclude pixels outside the desired range. A map of the excluded pixels is displayed after the range bars are moved.
In the Calibration window, click Save, name the table, and continue. (The file will have a .cal extension.)
Test the new calibration slide by moving it to a new area and collecting an image.
Look for patterns in the image that may have resulted from non-uniformities in the fluorescent field. If any occur, collect an image of yet another region, and verify that the inhomogeneity was in the second rather than the first field. The total range of calibrated intensities, from minimum to maximum, are typically less than about 10% of the mean.
If the calibration table is not acceptable, generate a new calibration table from a different part of the slide.