Intensity calibration and shading correction for fluorescence microscopes

Abstract

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.