Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry

Cytometry A. 2006 Mar;69(3):139-41. doi: 10.1002/cyto.a.20227.

Abstract

Background: Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities.

Methods: Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation.

Results: Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible.

Conclusion: The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD19 / analysis
  • CD3 Complex / analysis
  • CD5 Antigens / analysis
  • Fluorescent Dyes / chemistry*
  • Humans
  • Laser Scanning Cytometry / methods*
  • Leukocytes / chemistry
  • Leukocytes / cytology
  • Photobleaching*

Substances

  • Antigens, CD19
  • CD3 Complex
  • CD5 Antigens
  • Fluorescent Dyes