SUMMARYFluorimetric measurements with Ca2+‐chelating or pH‐sensitive dyes at the level of individual small cells (diameter<10 μm) require special modifications of and attachments to conventional light microscopes: (1) a subassembly for multiple‐wavelength excitation by rapidly interchanging narrow‐band interference filters in the excitation light path permits quasi‐continuous dual‐ or triple‐wavelength determinations of the signals as well as ratio‐forming; (2) a gated, photon‐counting multiplier tube is mandatory for ultrasensitive light measurements. Data processing is done digitally and accomplished by circuitry using standard logic elements, for example ECL, TTL and CMOS counters and gates.We also present examples of application to some typical problems in the field of cell biology: (A) distribution of Ca2+in isolated, living cells; (B) Ca2+‐transients in single cells and tissue explants exposed to selected stimuli.Outlines are given for converting conventional light microscopes to ultrasensitive photon‐counting analysing instruments which may then be modified according to the experimental requirements and the type of microscope available. In addition, we determined whether calibration techniques used in macroscopical work, i.e. with conventional spectrofluorimeters, also produced meaningful results when applied to microspectrofluorimetry of very small cells that yiel
展开▼
