Spectral overlap considerations for simultaneous measurement of intracellular calcium concentration and transmembrane potentials in cardiac tissue

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 has been used to measure intracellular calcium concentration and Di-4 ANEPPS has been used to measure membrane potentials in cardiac tissue. This paper addresses the feasibility of using these fluorescent dyes on the heart together and thereby measuring membrane potentials and intracellular calcium concentration simultaneously. In order for the dyes to be used simultaneously, it is necessary for their respective fluorescent spectra to be sufficiently separated in wavelength, thus allowing them to be separated by optical filters or spectrographs. An apparatus was constructed to measure the dyes' spectra. The spectra obtained from this equipment were mathematically modeled in order to assess the spectral overlap error under different conditions. Error curves were constructed which may guide the researcher to pick optical filters and dye concentrations that will result in an acceptable error.