Intrinsic Biomarker for Oxidative Stress by FLIM

Abstract

Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed single point Raman spectroscopy which revealed typical lipid droplet Raman-bands. LLS FLIM signature was also observed in mouse perigonadal white adipose tissue (WAT) lipid droplets, which co-localized with THG signal. This further confirms the association of LLS to lipid droplets. We show application of LLS as OS biomarker in cardiology and cancer. An increase of LLS was observed in induced-pluripotent-stem-cell-derived cardiomyocytes under OS due to hypoxia. This correlates to the increase in ROS production due to hypoxia. LLS FLIM signature was also observed in melanoma, a ROS driven cancer. LLS were mostly located in the dendritic projection of the melanoma cells. Comparatively, only a small amount of LLS was displayed by melanocytes. This label-free, non-invasive FLIM-phasor method is a promising imaging technique for OS detection in biological systems, especially for in vivo measurements.Work supported in part by NIH grants P50GM076516,P41GM103540 and UH2TR000481-0.