The optical properties of a confocal scanning microscope that was designed to utilize a synchrotron as light source are presented. The usable spectral range is from 200 nm up to 700 nm. Using 325-nm laser light, it is shown that the lateral resolution is about 125 nm, and the axial resolution better than 250 nm. After transport of the microscope from Utrecht to the Daresbury Synchrotron Source, 200-nm excitation can be applied, and the lateral resolution will drop to below 100 nm.

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.

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper.