Fluorescent dyes for shading correction

Since my recent post on shading correction of microscopy images, I’ve become aware of two papers by Michael Model describing the use of concentrated dye solutions for shading correction and intensity calibration of microscopes. The first paper [1] describes the testing of the solutions, while the second paper [2] provides recipes for green, red, and far-red calibration solutions. He finds that concentrated solutions (10% w/v fluorescein, for instance) perform best, and also identifies dyes that are highly water soluble and can be prepared at these high concentrations for measurement of shading images.

In particular, he recommends fluorescein for the correction of green images, rose bengal or acid fuchsin for red images (all available from Sigma-Aldrich), and acid blue 9 for far-red (Cy5 images).


  1. M.A. Model, and J.K. Burkhardt, "A standard for calibration and shading correction of a fluorescence microscope.", Cytometry, 2001. http://www.ncbi.nlm.nih.gov/pubmed/11500847
  2. M.A. Model, "Intensity calibration and shading correction for fluorescence microscopes.", Current protocols in cytometry, 2006. http://www.ncbi.nlm.nih.gov/pubmed/18770832