A HIGH-RESOLUTION, CONFOCAL LASER-SCANNING MICROSCOPE AND FLASH-PHOTOLYSIS SYSTEM FOR PHYSIOLOGICAL-STUDIES

We describe the construction of a high-resolution confocal laser-scann ing microscope, and illustrate its use for studying elementary Ca2+ si gnalling events in cells. An avalanche photodiode module and simple op tical path provide a high efficiency system for detection of fluoresce nce signals, allowing use of a small confocal aperture giving near dif fraction-limited spatial resolution (< 300 nm lateral and < 400 nm axi al). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibil ity to record line-scans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV i rradiation system permits simultaneous photolysis of caged compounds o ver either a uniform, wide field (are lamp source) or at a tightly foc ussed spot (frequency-tripled Nd:YAG laser). The microscope thus provi des a versatile tool for optical studies of dynamic cellular processes , as well as excellent resolution for morphological studies. The confo cal scanner can be added to virtually any inverted microscope for a co mponent cost that is only a small fraction of that of comparable comme rcial instruments, yet offers better performance and greater versatili ty.