Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences

Joseph R. Lakowicz; Ignacy Gryczynski; Henryk M. Malak; Michael L. Johnson; Gabor Laczko; Wieslaw M. Wiczk; Henryk Szmacinski; Jozef Kusba

doi:10.1117/12.44239

1 July 1991 Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences

Joseph R. Lakowicz, Ignacy Gryczynski, Henryk M. Malak, Michael L. Johnson, Gabor Laczko, Wieslaw M. Wiczk, Henryk Szmacinski, Jozef Kusba

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Proceedings Volume 1435, Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications; (1991) https://doi.org/10.1117/12.44239
Event: Optics, Electro-Optics, and Laser Applications in Science and Engineering, 1991, Los Angeles, CA, United States

Abstract

Measurements of time-resolved fluorescence are increasingly used for research in biophysics, biochemistry, cell biology and medicine. Advances in the technology of light sources and detectors are resulting in more reliable and/or advanced instrumentation, which is resulting in the expanding applications of fluorescence spectroscopy. Time-resolved measurements are often performed by direct measurements in the time-domain. In this article the authors describe the alternative method of frequency-domain fluorometry. The frequency-response of the emission to intensity-modulated excitation can be used to recover the time-dependent decay. Commercial instrumentation now allows measurements to an upper light modulation frequency limit of 200 MHz. This laboratory has developed second and third generation instruments which allows measurements to 2 GHz and subsequently to 10 GHz. The frequency-domain data from such instrumentation provides excellent resolution of picosecond decays of intensity and anisotropy. Additionally, the frequency-domain method appears to provide remarkable resolution of complex decays which are often observed for biochemical samples. In this article the authors describe this instrumentation and applications of this method. Examples are shown using probes with ps decay and correlation times, the intrinsic fluorescence of proteins, and the measurement of end-to-end diffusion in proteins and/or flexible molecules.

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Joseph R. Lakowicz, Ignacy Gryczynski, Henryk M. Malak, Michael L. Johnson, Gabor Laczko, Wieslaw M. Wiczk, Henryk Szmacinski, and Jozef Kusba "Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences", Proc. SPIE 1435, Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications, (1 July 1991); https://doi.org/10.1117/12.44239

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KEYWORDS

Modulation

Picosecond phenomena

Diffusion

Fluorescence spectroscopy

Phase shift keying

Luminescence

Microchannel plates

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