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Published on: March 26, 2019
Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry
Anja Mittag1, Dominik Lenz, József Bocsi
1Department of Pediatric Cardiology, Heart Center Leipzig GmbH, University of Leipzig, Leipzig, Germany.
Summary
This study introduces a sequential photobleaching method using Laser Scanning Cytometry to differentiate fluorochromes with similar spectra. This technique enhances information density in polychromatic cytometry by leveraging differences in photostability.
Area of Science:
- Biotechnology
- Cellular Biology
- Analytical Chemistry
Background:
- Slide-based cytometry is crucial for polychromatic investigations.
- Distinguishing fluorochromes with similar emission spectra is challenging.
- Laser Scanning Cytometry offers relocalization and merge features for fluorochrome differentiation.
Purpose of the Study:
- To develop a method for distinguishing fluorochromes with comparable emission spectra but different photostabilities.
- To enhance information density in single-cell analysis.
- To improve polychromatic cytometry techniques.
Main Methods:
- Blood specimens were stained with fluorochrome pairs (FITC/ALEXA488, PE/ALEXA532, APC/ALEXA633).
- Repeated laser excitation was used to induce photobleaching.
- Sequential photobleaching was applied to differentiate fluorochromes based on photostability.
Main Results:
- ALEXA dyes demonstrated superior photostability compared to conventional fluorochromes (FITC, PE, APC).
- Differentiation within fluorochrome pairs was achievable due to varying photostability.
- The method successfully distinguished fluorochromes with overlapping emission spectra.
Conclusions:
- Sequential photobleaching increases information density at the single-cell level.
- This method is a valuable addition to polychromatic cytometry.
- The technique allows for more precise cellular analysis by resolving spectrally similar fluorochromes.
