Measurement of intracellular Ca2+ changes using novel caged cyclic nucleotides and confocal laser scanning microscopy

The intention of this study is to explore the applicability of confocal mic roscopy in conjunction with the use of caged cyclic nucleotide derivatives. The methodological potential of UV laser confocal microscopy has been asse ssed. it is shown that illumination of a single cell or a small area of a s ingle cell is possible, whereby the intracellular Ca2+ signal is measured a t illuminated and non-illuminated cells. Such measurements do not have a hi gh time resolution because of the specific system parameters. However, with an N-2 pulse laser (not part of the standard microscope set-up), Ca2+ sign als with a time resolution of around 100 ms have been measured. This facili tates investigation of the kinetics of Ca2+ influx, Intracellular Ca2+ meas urements at HEK293 and sperm cells have been made here, For sperm cells the advantages of confocal microscopy an best evidenced in conjunction with th e use of caged cyclic nucleotides; a cyclic nucleotide-gated Ca2+ influx at the tail of these cells has thereby been demonstrated for the first time. (C) 1999 Elsevier Science S.A. All rights reserved.