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 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.