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.


CONCLUSIONS
The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.
Department of Pediatric Cardiology, Heart Center Leipzig GmbH, University of Leipzig, Leipzig, Germany.
Anja
A
Mittag
Dominik
D
Lenz
József
J
Bocsi
Ulrich
U
Sack
Andreas O H
AO
Gerstner
Attila
A
Tárnok
D018941:Antigens, CD19 / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D018956:CD5 Antigens / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods*; D007962:Leukocytes / Q000737:chemistry / Q000166:cytology; D038761:Photobleaching
D018941:Antigens, CD19; D017252:CD3 Complex; D018956:CD5 Antigens; D005456:Fluorescent Dyes