Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences (Keynote Paper)

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

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Scopus citations

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.

Original languageEnglish
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
PublisherPubl by Int Soc for Optical Engineering
Pages142-160
Number of pages19
ISBN (Print)0819405256
StatePublished - 1991
EventOptical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications - Los Angeles, CA, USA
Duration: 21 Jan 199123 Jan 1991

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume1435
ISSN (Print)0277-786X

Other

OtherOptical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications
CityLos Angeles, CA, USA
Period21/01/9123/01/91

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    Lakowicz, J. R., Gryczynski, I., Malak, H., Johnson, M. L., Laczko, G., Wiczk, W. M., Szmacinski, H., & Kusba, J. (1991). Frequency-domain fluorescence spectroscopy: instrumentation and applications to the biosciences (Keynote Paper). In Proceedings of SPIE - The International Society for Optical Engineering (pp. 142-160). (Proceedings of SPIE - The International Society for Optical Engineering; Vol. 1435). Publ by Int Soc for Optical Engineering.