Abstract
Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets.
Highlights
Fluorescence lifetime imaging of endogenous biomarker of oxidative stress Rupsa Datta[1], Alba Alfonso-Garcıa[2], Rachel Cinco3 & Enrico Gratton[1]
The NADH-fluorescence lifetime imaging (FLIM) cluster is chosen by the blue cursor on the phasor plot, and this selection corresponds to regions surrounding the lipid droplet: adipocyte cytoplasm, nuclei, and the extracellular regions
We present results showing autofluorescent long lifetime species (LLS) linked to products of lipid oxidation by reactive oxygen species (ROS), and potential biomarker for oxidative stress
Summary
Fluorescence lifetime imaging of endogenous biomarker of oxidative stress Rupsa Datta[1], Alba Alfonso-Garcıa[2], Rachel Cinco3 & Enrico Gratton[1]. Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We show label-free detection of oxidative stress by fluorescence lifetime measurement of intrinsic fluorescent species using multiphoton fluorescence microscopy. These species with granular appearance co-localize with lipid droplets. Multiphoton fluorescence microscopy (MPM) has been employed previously to perform label free fluorescence lifetime imaging (FLIM) of intrinsic fluorophores like reduced nicotinamide adenine dinucleotide (NADH), collagen, retinol, and retinoic acid[11,12]. The lifetime information shown in the phasor plot can be mapped back to the image to resolve the spatial location of these species
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