UNIT 10.14 Intensity Calibration and Shading Correction for Fluorescence Microscopes

  1. Michael A. Model

Published Online: 1 AUG 2006

DOI: 10.1002/0471142956.cy1014s37

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Model, M. A. 2006. Intensity Calibration and Shading Correction for Fluorescence Microscopes. Current Protocols in Cytometry. 37:10.14:10.14.1–10.14.7.

Author Information

  1. Kent State University, Kent, Ohio

Publication History

  1. Published Online: 1 AUG 2006
  2. Published Print: JUL 2006

This is not the most recent version of the article. View current version (1 APR 2014)


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.


  • fluorescence microscopy;
  • confocal microscopy;
  • standardization;
  • calibration;
  • shading correction;
  • sodium fluorescein;
  • Acid Fuschin;
  • Rose Bengal;
  • Acid Blue 9