Intensity Calibration and Shading Correction for Fluorescence Microscopes

Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated dye solution placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photobleaching. The technique has a number of practical features that make it inexpensive, reproducible, and straightforward. Descriptions are given for both wide‐field and confocal scanning fluorescence microscopy.

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