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 important measurements used to study the mechanisms of cardiac activity. Fluorescent dyes have been used to measure these separately but not simultaneously in cardiac tissue. Fluo-3 and Fluo-4 (a recently improved version of Fluo-3) have been used to measure changes in intracellular calcium concentration and Di-4 ANEPPS has been used to measure transmembrane potentials. This paper addresses the feasibility of using these fluorescent dyes together in order to measure transmembrane potentials and intracellular calcium concentration simultaneously. For the dyes to be used simultaneously, their respective fluorescence spectra must be sufficiently separated in wavelength in order to allow them to be separated by optical filters or spectrographs. An apparatus was constructed to measure the dyes' spectra in a fluorescence imaging chamber as well as in an isolated perfused rabbit heart. The measured spectra were mathematically modeled in order to assess the spectral overlap error under different conditions. Error graphs were constructed which may help researchers select optical filters and dye concentrations that will result in an acceptable error.