Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry.

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.