Advances in frequency-domain fluorometry, gigahertz instrumentation, time-dependent photomigration, and fluorescence lifetime imaging

Joseph R. Lakowicz, Ignacy Gryczynski, Henryk Szmacinski, Kazimierz Nowaczyk, Michael L. Johnson

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

2 Scopus citations

Abstract

During the past seven years, there have been remarkable advances in the frequency-domain method for measurement of time-resolved emission or light scattering. In this presentation we describe the recent extension of the frequency range to 10 GHz using a specially designed microchannel plate PMT. Experimental data will be shown for measurement of picosecond rotational diffusion and for sub-picosecond resolution of time delays. The resolution of ps to ns timescale processes is not obtained at the expense of sensitivity or is it shown by measurements on the intrinsic tryptophan emission from hemoglobin. We also describe a time- resolved reflectance imaging experiment on a scattering medium containing an absorbing object. Time-resolved imaging of the back-scattered light is realized by means of a RF-phase- sensitive camera, synchronized to the laser pulses. By processing the stored images, a final image can be created, the contrast of which is based only on time differences of the back- scattered photons. This image reveals the presence and position of the absorber within the scattering medium. And finally, we describe a new methodology, fluorescence lifetime imaging (FLIM), in which the contrast depends on the fluorescence lifetime at each point in a two-dimensional image, and not the local concentration and/or intensity of the fluorophore. We used FLIM to create lifetime images of NADH when free in solution and when bound to malate dehydrogenase. FLIM has numerous potential applications in cell biology and imaging.

Original languageEnglish
Title of host publicationProceedings of SPIE - The International Society for Optical Engineering
PublisherPubl by Int Soc for Optical Engineering
Pages227-243
Number of pages17
ISBN (Print)0819407305, 9780819407306
DOIs
StatePublished - 1992
EventRecent Advances in the Uses of Light in Physics, Chemistry, Engineering, and Medicine - New York, NY, USA
Duration: 19 Jun 199121 Jun 1991

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume1599
ISSN (Print)0277-786X

Other

OtherRecent Advances in the Uses of Light in Physics, Chemistry, Engineering, and Medicine
CityNew York, NY, USA
Period19/06/9121/06/91

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    Lakowicz, J. R., Gryczynski, I., Szmacinski, H., Nowaczyk, K., & Johnson, M. L. (1992). Advances in frequency-domain fluorometry, gigahertz instrumentation, time-dependent photomigration, and fluorescence lifetime imaging. In Proceedings of SPIE - The International Society for Optical Engineering (pp. 227-243). (Proceedings of SPIE - The International Society for Optical Engineering; Vol. 1599). Publ by Int Soc for Optical Engineering. https://doi.org/10.1117/12.56701