Artifacts and diagnostics in fast fluorescence measurements

Gary R. Holtom

doi:10.1117/12.17680

1 May 1990 Artifacts and diagnostics in fast fluorescence measurements

Gary R. Holtom

Proceedings Volume 1204, Time-Resolved Laser Spectroscopy in Biochemistry II; (1990) https://doi.org/10.1117/12.17680
Event: OE/LASE '90, 1990, Los Angeles, CA, United States

Abstract

The goal of time resolved fluorescence experiments is to determine the validity of a model for population (kinetics) or motion (decay of anisotropy), and to extract a suitable parameter set which quantitatively describes the sample. Ideally, this analysis requires no adjustments of an ad-hoc nature in order to obtain a good fit, and the time-resolved residuals will be uniformly random. Any problems at this point lead to questions about the model being used, either with respect to its correctness in functional form, or in the exact values recovered, and it may become difficult to extract useful information about the sample. Reaching the ideal situation, and confirming that there are no experimental problems, requires considerable care. All aspects of the hardware are examined, starting with the laser light source and sample illumination optics. Collection optics for the fluorescence are analyzed, along with polarization components. Electronics checks and optimizations are described, showing the effects of characteristic problems. Finally, apparent artifacts due to numerical analysis are shown. While the examples are given for time-correlated single-photon counting, many of the optics related problems have similar consequences in the frequency domain. Some of the experimental problems have implications for the design of multiple channel detection schemes.

Citation Download Citation

Gary R. Holtom "Artifacts and diagnostics in fast fluorescence measurements", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); https://doi.org/10.1117/12.17680

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