We describe a fast and simple method to monitor variations of intracytoplasmic calcium concentrations in very small and motile cells such as mammalian spermatozoa during measurement time. The method combines a procedure of sperm cells semi-immobilization with microspectrofluorimetric measurement of intracytoplasmic calcium concentrations supported by videoimaging that allows also a constant monitoring of viability during the time of calcium recording. In this paper we show that a semi-immobilization of individual viable spermatozoa can be obtained preparing the agar matrix in a two step procedure. The cell immobilizing system proposed, associated with the microspectrofluorimetric analysis supported by videoimaging, is a simple, rapid and useful tool for those studies having the goal of correlating the presence and cellular distribution of ion channels with their functional status and their response to physiologic and/or pharmacological molecules.
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