Errors caused by combination of Di-4 ANEPPS and Fluo3/4 for simultaneous measurements of transmembrane potentials and intracellular calcium

Intracellular calcium concentration and transmembrane potentials are two im portant measurements used to study the mechanisms of cardiac activity. Fluo rescent dyes have been used to measure these separately but not simultaneou sly in cardiac tissue. Fluo-3 and Fluo-4 (a recently improved version of Fl uo-3) have been used to measure changes in intracellular calcium concentrat ion and Di-4 ANEPPS has been used to measure transmembrane potentials. This paper addresses the feasibility of using these fluorescent dyes together i n order to measure transmembrane potentials and intracellular calcium conce ntration simultaneously. For the dyes to be used simultaneously, their resp ective fluorescence spectra must be sufficiently separated in wavelength in order to allow them to be separated by optical filters or spectrographs. A n apparatus was constructed to measure the dyes' spectra in a fluorescence imaging chamber as well as in an isolated perfused rabbit heart. The measur ed spectra were mathematically modeled in order to assess the spectral over lap error under different conditions. Error graphs were constructed which m ay help researchers select optical filters and dye concentrations that will result in an acceptable error. (C) 1999 Biomedical Engineering Society. [S 0090-6964(99)01104-2].