Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis

Two-photon molecular excitation microscopy has several advantages over conv entional confocal fluorescence microscopy, including the ability to section deeper into scattering samples and to allow spatially resolved flash photo lysis. We describe and examine the benefit of incorporating non-descanned f luorescence detection in our microscope system. In a scattering sample wher e almost no signal could be obtained at a depth of 50 mu m with confocal de tection, non-descanned detection resulted in an improvement of signal stren gth by more than an order of magnitude at depths >40 mu m. The spatio-tempo ral properties of stationary spot two-photon excited flash photolysis (TPEF P) in drops of test solutions and cardiac myocytes were also examined. At i nput powers that produce >10% of the maximum rate of DM-nitrophen photolysi s, serious photodestruction of the reporter fluorochrome (Fluo-3) at the ph otolysis spot occurred. At power levels of similar to 4 mW for periods <50 ms, we were able to produce small repeatable calcium release events using D M-nitrophen in cardiac myocytes, which were similar to naturally occurring calcium sparks. The properties of these artifical calcium sparks were very similar to signals obtained from drops of test solutions, suggesting that t he apparent rate of calcium diffusion in myocytes is similar to the rate of diffusion of Fluo-3 in solution. Using TPEFP, we also examined the ability of a combination of EGTA and a low-affinity calcium indicator to track the time course of calcium release. Although the addition of EGTA improved the temporal fidelity of the rise of the calcium signal, it did not significan tly reduce the spread of the fluorescence signal from the photolysis spot. (C) 1999 Wiley-Liss, Inc.