Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry

Authors


  • Part of this work was presented at the 10th Leipziger Workshop “Systems Biology and Clinical Cytomics,” April 7–9, 2005, Leipzig, Germany.

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. © 2006 International Society for Analytical Cytology

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