Elimination of light scatter interference in dual-laser flow cytometry by synchronous detection of emitted fluorescence: theory and demonstration using simulated signals

John A. Steinkamp; Jan F. Keij

doi:10.1117/12.349198

1 June 1999 Elimination of light scatter interference in dual-laser flow cytometry by synchronous detection of emitted fluorescence: theory and demonstration using simulated signals

John A. Steinkamp, Jan F. Keij

Author Affiliations +

Proceedings Volume 3604, Optical Diagnostics of Living Cells II; (1999) https://doi.org/10.1117/12.349198
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

Light scatter is often a source of interference in dual-laser excitation experiments by flow cytometry. If the two laser beams are not adequately separated or optical masking is not used, and if gated sequential fluorescence signal detection is not employed, light scatter from particles/cells as they pass across one beam can interfere in the fluorescence measurement channel of the other laser excitation beam. In this study we discuss the problem and present the theory for improving fluorescence measurements in dual-laser flow cytometry by use of modulated laser excitation and synchronous (phase- sensitive) detection of the resulting fluorescence emission to eliminate light scatter interference from particles/cells. Fluorescence emission from particles/cells generated by the first laser beam (unmodulated cw) is measured using conventional means (detection channel no. 1). Light scatter interference from particles/cells as they pass across the first laser excitation beam, whose wavelength lies in the fluorescence measurement region of detection channel no. 2, is rejected by the synchronously tuned phase-sensitive channel no. 2 detector, whereas the fluorescence emission corresponding to laser no. 2 excitation is detected and measured. The efficacy of the technology in relation to analytical cytology measurements is demonstrated using simulated fluorescence and light scatter signals.

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John A. Steinkamp and Jan F. Keij "Elimination of light scatter interference in dual-laser flow cytometry by synchronous detection of emitted fluorescence: theory and demonstration using simulated signals", Proc. SPIE 3604, Optical Diagnostics of Living Cells II, (1 June 1999); https://doi.org/10.1117/12.349198

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