Abstract

Time-resolved autofluorescence spectroscopy permits characterization of biological tissues and has already shown the potential in a number of disease applications. It harnesses the photo-physical properties of a number of endogenous molecules, e.g. NADH and flavoproteins, to offer an opportunity to discern the energetic state of the myocardium without the issues of toxicity or consequences associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules, e.g. collagen, can provide information on structural changes to the heart. We report the development and application of a custom fibre-optic probe-based time-resolved spectrofluorometer combining time-correlated single photon counting detection and white-light reflectometry to characterize the autofluorescence ‘fingerprint’ associated with the changes in histology, morphology, metabolism and function in heart health and pathological states. Measurement of fluorescence lifetime is inherently ratiometric and unaffected by molecule concentration and excitation intensity amongst other experimental variables. Furthermore fluorescence lifetime is affected by changes in the microenvironment e.g. protein binding states. Explanted hearts from male SD rats perfused using the ex vivo Langendorff system demonstrated stable fluorescence lifetimes. Fluorescence lifetime signals were successfully derived from fluorescence decays with acquisition times as low as 1 second. The changes in the autofluorescence signal of ex vivo Langendorff preparations using perfusate column switching were characterised for an ischaemia-reperfusion model and for a change of substrate between glucose and mannitol. This technology has the potential to be a label-free method to observe biochemical changes associated with disease mechanisms in the heart, with clear potential application to cardiac research and clinical diagnostics.