Search

We have developed a stroboscopic near-field optical microscope for observation of biological specimens and observed glycerinated muscles before and after muscle contraction with the developed system. In the system, the optical field distribution localized near the specimen is recorded as the surface topographic distribution of a photosensitive film surface. Our system is very useful for observing living biological specimens with high resolutions, because it is possible to get stroboscopic image by using a photosensitive film as detecting optical distributions instead of a scanning of probes. We have succeeded in observing inner structures of muscle cells with sub-wavelength resolution and achieved higher contrast than an ordinary optical microscope.

Spiral wave phenomena exist in many scales of nature and have attracted the attention of scientists from different fields. Although much work has been done on qualitative analysis of spiral waves, the mechanism of spiral waves' spontaneous formation and termination in living systems is still not clear. Here, by using total internal reflection fluorescence microscopy, we show the spiral waves of calcium signals in single rat cardiac myocytes and the simulation of the waves comprising calcium sparks. The mechanism of the formation and termination of spiral waves is attributable to the calcium release channels' refractory resulting from their stochastic release. We suggest that this mechanism can be adapted to other living systems.

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two- to eight-channel, time-gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time-gates are significantly more sensitive than the two time-gate system. Only minor sensitivity differences were found between systems with four or more time-gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP-PMTs and 300-400 ps for other detectors. The maximum count rate of fast MCP-PMTs, however, is 10-100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP-PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.