CONSULTATIONS ON APPLICATIONS OF MULTI PHOTON MICROSCOPY

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A technique for the measurement of the two-photon uncaging action cross section of calcium cages has been developed in this laboratory $^{1,2}$. Using this technique, we report on the prognosis for effective two-photon excitation of a novel calcium cage, DMNPE-4 $^{3}$. We also describe calculations which predict the total amount of calcium ions uncaged and the temporal behavior of the liberated calcium distribution under given multiphoton photolysis conditions with a given cage. These calculations, combined with the two-photon uncaging action cross sections that we have measured for a variety of calcium cages enable the quantitative study of calcium dependent processes with high spatial resolution within cells or thick tissue samples. The ultimate intent of these studies is the application of these tools to the quantitative study of the spontaneous transient calcium concentration elevations ("sparks") in developing skeletal muscle cells, probing the potentiation/inhibition relation ships between consecutive sparks in one localized region as well as between sparks in spatially separate regions. 1. Shear, J. et al. {\em Biophys. J.}70(2) A211 (1996) 2. Brown et al, Manuscript in Preparation (1997) 3. Ellis-Davies, G.{\em Biophys. J.}72(2) A246 (1997)