Intensity calibration and shading correction for fluorescence microscopes

Curr Protoc Cytom. 2006 Aug:Chapter 10:Unit10.14. doi: 10.1002/0471142956.cy1014s37.

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.

MeSH terms

  • Calibration
  • Fluorescein
  • Image Enhancement / methods
  • Image Interpretation, Computer-Assisted / methods
  • Image Interpretation, Computer-Assisted / standards
  • Image Processing, Computer-Assisted / methods
  • Microscopy, Confocal / methods*
  • Microscopy, Confocal / standards
  • Microscopy, Fluorescence / methods*
  • Microscopy, Fluorescence / standards
  • Sensitivity and Specificity

Substances

  • Fluorescein