Micro-Volume Time-Resolved Fluorescence Spectroscopy Using a Confocal Synchrotron Radiation Microscope

Abstract

The confocal microscope SYCLOPS (SYCLOPS is not an acronym although the first syllable is derived from its main light source, a synchrotron radiation source) at the Daresbury Science and Engineering Research Council (SERC) Laboratory, U.K., has been designed to facilitate fluorescence decay measurements on microscopic volume elements. SYCLOPS utilizes the Daresbury Synchrotron Radiation Source (SRS) as a pulsed light source. Visible or UV excitation light is selected from the white synchrotron radiation spectrum with a bandpass filter matching the absorption band of the fluorophores. Decay curves of fluorescence intensity emitted by a pre-selected micro-volume in the sample can be acquired with standard time-correlated single-photon counting techniques. The fluorescence intensity was collected from a confocal spot with a volume of 3 μm³. The first lifetime measurements done with the instrument were carried out on a coumestrol l]benzopyran-6-one) solution and on different cellular compartments of living cells incubated with coumestrol.

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