MICROVOLUME TIME-RESOLVED FLUORESCENCE SPECTROSCOPY USING A CONFOCAL SYNCHROTRON-RADIATION MICROSCOPE

The confocal microscope SYCLOPS (SYCLOPS is not an acronym although th e first syllable is derived from its main light source, a synchrotron radiation source) at the Daresbury Science and Engineering Research Co uncil (SERC) Laboratory, U.K., has been designed to facilitate fluores cence decay measurements on microscopic volume elements. SYCLOPS utili zes the Daresbury Synchrotron Radiation Source (SRS) as a pulsed light source. Visible or UV excitation light is selected from the white syn chrotron radiation spectrum with a bandpass filter matching the absorp tion band of the fluorophores. Decay curves of fluorescence intensity emitted by a pre-selected micro-volume in the sample can be acquired w ith standard time-correlated single-photon counting techniques. The fl uorescence intensity was collected from a confocal spot with a volume of 3 mu m(3). The first lifetime measurements done with the instrument were carried out on a coumestrol -dihydroxy-6H-benzofuro[3,2-c][I]ben zopyran-6-one) solution and on different cellular compartments of livi ng cells incubated with coumestrol.