A Time Resolved Fluorescence Spectrometer with Sub-Millisecond Data Acquisition Time
| Autor: | Ewa Prochniewicz, Greg Gillispie, David J. Kast, Igor V. Negrashov, Joseph M. Muretta, David D. Thomas, Yuri E. Nesmelov, Roman V. Agafonov, Piyali Guhathakurta |
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| Rok vydání: | 2010 |
| Předmět: |
0303 health sciences
Millisecond Spectrometer business.industry Chemistry Biophysics Molecular physics Fluorescence 03 medical and health sciences 0302 clinical medicine Optics Förster resonance energy transfer Data acquisition biological sciences Transient (oscillation) Time-resolved spectroscopy business Laser-induced fluorescence 030217 neurology & neurosurgery 030304 developmental biology |
| Zdroj: | Biophysical Journal. 98:585a-586a |
| ISSN: | 0006-3495 |
| Popis: | We have developed a high-throughput time-resolved fluorescence spectrometer capable of recording a high-resolution time-domain (sub-nanosecond) fluorescence decay, with high S/N every 100 μs. Coupled with a conventional stopped-flow rapid mixer, this technology has allowed us to measure changes in time-resolved fluorescence decays occurring during the course of a millisecond-resolved biochemical transient experiment. Most instruments used in fluorescence-based kinetic studies are limited to detecting a single fluorescence intensity signal on the millisecond time scale. While this type of fluorescence intensity-based measurement is informative, it provides scant information compared to a full sub-nanosecond resolved fluorescence decay- which is exquisitely sensitive to the structure, dynamics, and interactions of molecules in the sample. Current fluorescence lifetime technology is too slow by a factor of at least 1000 to detect this decay accurately within the millisecond timescale of a typical biochemical transient. Our system, based on a direct acquisition data collection approach, records the entire fluorescence decay with S/N > 100 from a single pulse form a 10 kHz microlaser. Using this approach, we can resolve individual fluorescence lifetime components comprising as little as 10% of a complex multi-exponential fluorescence decay. When used to monitor structural transitions by time resolved FRET, we are capable of isolating individual structural states within complex structural ensembles. Using this approach we were able to simultaneously monitor the binding of myosin to different classes of binding sites on actin filaments. This type of analysis is not possible with conventional fluorescence based stopped flow measurements. |
| Databáze: | OpenAIRE |
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