Multiplexed Fiber-Optic Fluorescence for Functional Monitoring of Perfused Hearts

Monitoring molecular dynamics in the heart is essential for advancing our understanding of cardiac physiology and biochemistry in both healthy and diseased states, as well as for guiding the development and evaluation of novel cardiac therapies. We present a multiexcitation, ratiometric fiber-optic spectroscopic platform for noninvasive, real-time monitoring of biochemical and physiological processes in isolated Langendorff-perfused rat hearts. The system employs a fiber-optic balloon probe capable of concurrent optical measurements and intraventricular pressure sensing, thereby providing complementary physiological data. A multiedge bandpass filter enables parallel fluorescence spectroscopy, allowing simultaneous detection and analysis of both exogenous and endogenous fluorophores. Coupled with multivariate regression analysis, we demonstrate the accurate quantification of fluorophore concentrations, facilitating comprehensive assessment of cardiac biochemical and functional dynamics. To mitigate geometric variability and motion artifacts, we developed a robust ratiometric approach using paired fluorescence agents. We demonstrate the system's capability by employing the fluorescent lipophilic cation tetramethylrhodamine ethyl ester (TMRE) as a noninvasive biomarker for mitochondrial membrane potential, extracting physiologically relevant metrics. This platform enables sensitive assessment of cardiac function with established fluorescent probes and holds promising potential as a versatile tool for investigating the dynamics of novel fluorophores.