TY - GEN
T1 - Advances in frequency-domain fluorometry, gigahertz instrumentation, time-dependent photomigration, and fluorescence lifetime imaging
AU - Lakowicz, Joseph R.
AU - Gryczynski, Ignacy
AU - Szmacinski, Henryk
AU - Nowaczyk, Kazimierz
AU - Johnson, Michael L.
PY - 1992
Y1 - 1992
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026737066&partnerID=8YFLogxK
U2 - 10.1117/12.56701
DO - 10.1117/12.56701
M3 - Conference contribution
AN - SCOPUS:0026737066
SN - 0819407305
SN - 9780819407306
T3 - Proceedings of SPIE - The International Society for Optical Engineering
SP - 227
EP - 243
BT - Proceedings of SPIE - The International Society for Optical Engineering
PB - Publ by Int Soc for Optical Engineering
T2 - Recent Advances in the Uses of Light in Physics, Chemistry, Engineering, and Medicine
Y2 - 19 June 1991 through 21 June 1991
ER -