1 May 1990 48000 MHFTM: a dual-domain Fourier transform fluorescence lifetime spectrofluorometer
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Abstract
The technical simplicity and speed of cross-correlation frequency domain fluorometry has been compromised recently by the need to measure and analyse the phase and modulation responses of a sample over a wide range of excitation frequencies. Phase fluorometers currently use single frequency cross-correlation detection combined with either single frequency excitation or harmonic excitation from a pulsed source. Phase and modulation response curves are constructed by taking a series of point-by-point measurements over the frequency region of interest. This can be a time consuming process which requires the attention of an experienced operator. The Fourier transform spectrofluorometer described here combines multi-harmonic excitation with multi-harmonic cross-correlation detection and digital signal processing techniques to implement a frequency domain fluorometer which can acquire phase and modulation responses simultaneously at 40 to 50frequencies. A particularly dramatic capability of the Fourier transform fluorometer is its ability to collect multi-frequency data at millisecond rates thus allowing complex lifetime analysis of rapidly evolving fluorescence systems. Further, multi-harmonic excitation and detection is the equivalent of measuring the time course of a sample's response to impulse excitation. Therefore, time domain data is inherently available from the instrument and may be fitted to models of fluorescence decay by iterative reconvolution techniques as an alternative non-linear least squares fitting of the frequency domain data.
© (1990) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
George Weston Mitchell and Kerry M. Swift "48000 MHFTM: a dual-domain Fourier transform fluorescence lifetime spectrofluorometer", Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17770; https://doi.org/10.1117/12.17770
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