Measuring fluorescence decay times by phase-shift and modulation techniques using the high harmonic content of pulsed light sources

E Gratton,R Lopez-Delgado

doi:10.1007/bf02738361

Abstract

In the present work we discuss the possibility to measure fluorescence lifetimes by coupling pulsed excitation with phase-shift and amplitude-modulation detection techniques. We show that, because of the high harmonic content of narrow light pulses, the sample may be considered as simultaneously excited with a set of modulation frequencies up to the GHz region with noticeable power. By measuring then the phase-shift and modulation ratio of fluorescence with respect to the exciting light in the high-frequency region, impressive time resolutions may be achieved. On the other hand, since we dispose of a wide range of modulation frequencies, the problem of the multiexponential decay may be easily handled and has an exact analytical solution, provided the signal-to-noise ratio is good enough. We anticipate the possibility to introduce cross-correlation methods in order to perform the experiment in the very-low-frequency region. A short discussion about the notion of time resolution for this kind of measurement is also included.

Highlights

Atomic and molecular excited states can deactivate through a variety of relaxation channels, one of them being the radiative one
COUPLING PULSE/) EXCITATION WITH PHASE AND MODULATION METHODS. With this new method we show in table I that impressive time resolution may be achieved with, for example, CW mode-locked lasers and it becomes clear that at this level of resolution the optical ~nd geometrical characteristics of the experiment have to be carefully designed, since a difference of,~ 0.3 mm on two light paths will already introduce a phase shift corresponding to --~ 1 ps
With ordinary readily available phase-sensitive devices phases may be measured with 0.1 degree accuracy, and in table I we show the ultimate time resolution (Ave) achieved in the fluorescence lifetime measurement with some typical pulsed light sources when the phase shift is monitored with the harmonic component whose intensity in the power spectrum is about 10 % of that of the fundamental

Summary

Introduction

Atomic and molecular excited states can deactivate through a variety of relaxation channels, one of them being the radiative one. This emission of light (called in general luminescence), and its time properties,. Luminescence phenomena span through an impressive number of orders of magnitude (from many seconds, as in phosphorescence from metastable excited states, to as short as J0 -'~ s or less, as in X-ray fluorescence), and it is easy to understand the interest to obtain radiative decay rate with great accuracy as well as to be able to measure very short fluorescence lifetimes in order to check theoretical models on molecular relaxation (photophysical and photochemical) processes. Time-resolved spectrofluorometry is becoming an important and widespread tool in biophysics and biochemistry

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Conclusion