Two dye two wavelength excitation calcium imaging: results from bovine adrenal chromaffin cells.
We tested a mixture of Calcium-Green-1 (CG-1) and Brilliantsulfaflavine (BS) for dual excitation ratiometric measurements of the intracellular free calcium concentration ([Ca2+]i) in bovine adrenal chromaffin cells. Dyes were coloaded (without being molecularly linked to each other) 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 photobleaching. 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 signals could be derived using a Grynkiewicz-type equation; (ii) BS is an ideal reference dye that displayed no interaction with CG-1 or cellular constituents; and (iii) that calibration requires diffusional equilibration between pipette and the accessible volume of the cell. Spatially resolved recordings of fluorescence excitation spectra revealed elevated fluorescence of CG-1 in the nucleus such that reported [Ca2+]i levels seemed 25% higher compared to cytosolic values. Comparing fluorescence emission from in vitro dye solutions with in vivo values, we could estimate the accessible volume fraction and amount of Ca(2+)-insensitive dye.
Oheim
M
M
Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany. oheim@mail.mpiem.gwdg.de
Naraghi
M
M
Müller
T H
TH
Neher
E
E
D000311:Adrenal Glands / Q000737:chemistry* / Q000166:cytology; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002417:Cattle; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D019439:Chromaffin Cells / Q000737:chemistry*; D003911:Dextrans; D005456:Fluorescent Dyes; D007546:Isoquinolines; D009930:Organic Chemicals; D012016:Reference Values; D013057:Spectrum Analysis
D003911:Dextrans; D005456:Fluorescent Dyes; D007546:Isoquinolines; D009930:Organic Chemicals; C071336:calcium green; C026796:brilliant sulfaflavine; D002118:Calcium