Application of real-time confocal microscopy for observation of living cells in tissue specimens

Measurement of intracellular Ca2+ concentration ([Ca2+]i) has been a fundamental technique in cell biology. However, most investigations have used cultured or isolated cells as an experimental model, and consequently can provide only limited insight into the mechanisms that operate in tissue in situ. Useful information may be obtained by studying intact tissue specimens. High-speed confocal microscopes that can acquire digital images at video rate have recently been developed. These confocal microscopes which can acquire data in real-time enable [Ca2+]i dynamics of individual cells in intact tissue specimens to be observed. The present paper examines the use of fluorescent microscopy and confocal microscopy for [Ca2+]i imaging of living tissue. We analyzed the dynamics of the duodenal gland, lacrimal gland, intestinal smooth muscles, arterioles, myenteric plexus, and dorsal root ganglion. In these specimens, individual cells exhibited different [Ca2+]i dynamics, and the responses to transmitters/modulators were heterogeneous. In conclusion, real-time imaging provides a useful tool for observing dynamic changes in cells in situ, and it may lead to improve understanding tissue physiology.