2 DYE 2 WAVELENGTH EXCITATION CALCIUM IMAGING - RESULTS FROM BOVINE ADRENAL CHROMAFFIN CELLS

We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavin e (BS) for dual excitation ratiometric measurements of the intracellul ar free calcium concentration ([Ca2+](i)) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each o ther) in the whole-cell configuration of the patch clamp technique. We compared the loading time-courses of CG-1 and BS, investigated their intracellular distribution patterns and studied the time course of pho tobleaching. We determined the apparent dissociation constant of CG-1, both optically and by potentiometric titration, Our findings indicate that: (i) with excitation at 420/488 nm, calibrated fluorescence sign als could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellula r constituents; and (iii) that calibration requires diffusional equili bration between pipette and the accessible volume of the cell. Spatial ly resolved recordings of fluorescence excitation spectra revealed ele vated fluorescence of CG-1 in the nucleus such that reported [Ca2+](i) levels seemed 25% higher compared to cytosolic values. Comparing fluo rescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca2+-insen sitive dye.