Gigahertz Frequency-Domain Fluorometry: Resolution of Complex Intensity Decays, Picosecond Processes and Future Developments
In this article we describe the principles, instrumentation and applications of frequency-domain fluorescence spectroscopy. This method can be used to evaluate complex time-dependent processes, even when the characteristic times are below 1 ns. This method is hence complementary to time-correlated single photon counting, in that one measures the frequency-response of the emission to modulated excitation, instead of the time response of the emission to pulsed excitation. To date, there have been relatively few attempts to measure photon migration in tissues by the frequency-domain method. We will demonstrate its potential for such measurements by demonstrating the resolution of multi-exponential decays on the psec timescale. The present instrumentation allows measurements to 2 GHz, which is easily adequate for measuring decay times as short as 50 ps. In the future it should be possible to extend the frequency range to 4 GHz, which should allow still faster processes to be quantified. Also, we have performed preliminary experiments in strongly scattering solutions which indicate that the phase shifts of the scattered light depend upon the amount of light absorption by the sample. It should be noted that this resolution of fast processes is not obtained at the expense of loosing information on the slower processes. Additionally, the GHz frequency-domain measurements are performed using low excitation intensities which do not damage the samples.
KeywordsDecay Time Rose Bengal Microchannel Plate Harmonic Content Fluorescence Decay Kinetic
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