An epi-fluorescent spinning-disk confocal microscope

Abstract

The confocal microscope offers a number of advantages over the conventional light microscope in improvement in spatial resolution optical sectioning by rejecting out-of-focus signals. The applications of confocal microscopy in biology will be greatly expanded if real-time imaging can be achieved.A real-time epi-fluorescent confocal microscope based on a spinning disk design has been constructed at our laboratory (Fig. 1). Different from Petran's tandem scanning configuration, but similar to Kino's, our microscope has both the illumination and the detection rays sharing the same pinholes (Fig. 2). As a result, the optical arrangement is simpler and much easier to align. The spinning “Nipkow disk” used in our microscope has 194,400 pinholes (25х25μm) arranged in 90 interleaved spirals (Fig. 3). The minimum and maximum spacing between adjacent pinholes are 90μm and 207μm respectively. When the disk is spun at 1000rpm, we achieved a frame rate of 1500f/sec with 1440 lines/frame. The disk is spun by a simple mechanism mounted on a X-Y linear slide (Fig. 4). The disk was fabricated by lithography on a 4 inch glass substrate (Fig. 5). At the present time, a 5W Argon ion laser or a 200W high pressure mercury arc lamp is used as the light source. By a set of mechanical slides, the microscope operates not only in epi-fluorescent and reflective confocal modes, but also in conventional epi-fluorescent and transmitted modes (i.e. bright field, phase contrast etc.). The low intensity confocal fluorescent image is amplified by the use of a TV-coupled second generation image intensifier. Then, the TV signal from the video camera is digitized and processed by a frame store interfaced to a 80386 microcomputer. In addition, the fine focus control of the microscope is driven by a stepping motor via the same computer.