PHYSICS AND CHEMISTRY OF PHOTORECEPTION

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Calcium-sensitive dyes, FLO-3 and Fura-2 have been introduced into retinal rods of living frog retinas by hydrolytic transfer of lipid-soluble esters. Using intensified video microscopy and quantitative image analysis, responses of intact rod cells and of isolated outer segments to changes in external calcium ion activity have been followed in order to study calcium regulation in these cells. The hydrolysis of lipid-soluble esters of the indicator dyes is very non-uniform from cell to cell, and two classes of rods are observed: one type constituting 12% of all rods accumulating 5-10 times as much indicator as the other 98-99% of the rod population within a given incubation period. The two populations can be demonstrated with a variety of indicators esterified with acetoxymethyl groups. Calcium indicators in the heavily labeled cells respond only weakly to changes in external calcium activity unless the plasma membranes have been made leaky with calcium-bearing ionophores. New pyroelectric detectors with stacked PVDF films have been prepared to increase heat sensitivity of the devices and to reject acoustic pickup. A recording amplifier that compensates for sudden heating and cooling effects caused changing the bathing solutions around tissues on the calorimeter detector has been designed and is under construction. Extension of pyroelectric methods to solution microcalorimetry is under way. 4(5) bromoimidazole has been tested as a marker for the aqueous cytoplasmic spaces in cells that are later freeze-dried for electron-probe microanalysis. Using a variety of water-loaded dextran polymers as test systems, the accuracy of estimating water content by analyzing residual Br content of the dried polymers has been confirmed. Water content of the various components of living retinas has been estimated and shown to agree with other, less selective methods. Bromoimidazole should be useful as a water marker in many other types of tissues.