Multiemission wavelength picosecond time-resolved fluorescence decay data obtained on the millisecond time scale: application to protein--DNA interactions and protein-folding reactions

Joseph M. Beechem

doi:10.1117/12.58264

1 April 1992 Multiemission wavelength picosecond time-resolved fluorescence decay data obtained on the millisecond time scale: application to protein:DNA interactions and protein-folding reactions

Joseph M. Beechem

Author Affiliations +

Proceedings Volume 1640, Time-Resolved Laser Spectroscopy in Biochemistry III; (1992) https://doi.org/10.1117/12.58264
Event: OE/LASE '92, 1992, Los Angeles, CA, United States

Abstract

One of the major aspects of fluorescence spectroscopy which differentiates this technique from many other spectroscopic approaches is the inherent multidimensional nature of the data. For instance, the basic pulsed-laser fluorescence data set is characterized by fluorescence versus: emission wavelength, polarization state (parallel and perpendicular intensities), time of emission (picoseconds to nanoseconds), and time of biological reaction (milliseconds to minutes). Usually, this six-dimensional data set is obtained piecemeal, single dimension at a time; often complete data sets are not even collected. This is especially true of the biological time scale axis. Data acquisition times for picosecond decay data are typically seconds to minutes, and, therefore, it has not been generally possible to perform this experiment in a kinetic mode. What is described in this report is the construction of a parallel multichannel time-correlated single-photon counting (TCSPC) fluorometer which is capable of simultaneous collection of: fluorescence vs. picosecond to nanosecond time vs. emission wavelength vs. polarization state vs. millisecond to second time. Use is made of two multi-anode microchannel plate detectors, each obtaining data at two different polarization states, six different emission wavelengths, along 12 independent TCSPC channels. This instrument is interfaced to a three-syringe stepper motor controlled stop-flow apparatus, and picosecond decay data along all of these channels is stored and collected by two 33 MHz 80486 computers at rates approaching 1200 - 12000 data sets per second.

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Joseph M. Beechem "Multiemission wavelength picosecond time-resolved fluorescence decay data obtained on the millisecond time scale: application to protein:DNA interactions and protein-folding reactions", Proc. SPIE 1640, Time-Resolved Laser Spectroscopy in Biochemistry III, (1 April 1992); https://doi.org/10.1117/12.58264

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