Study of cellular metabolism and its influence on physiological functions and pathology along with investigation of oxidative stress in pathogenesis are essential for fundamental biology as well as biomedical research. Optical imaging offers the opportunity to assess these indices non-invasively. In this work we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic and oxidative stress imaging in a wide range of biological samples. Analysis of FLIM data was performed by applying the ‘fit-free’ phasor approach where each pixel of the image is transformed to its corresponding phasor on the phasor plot. Biological systems are a rich resource of autofluorescent biomolecules. Their fluorescence lifetimes are sensitive to alteration of normal physiology, making them attractive endogenous probes. We discovered one such endogenous fluorophore with characteristic long fluorescent lifetime. We hypothesized these long lifetime species (LLS) to be fluorescent products of lipid oxidation by reactive oxygen species (ROS), rendering them biomarkers of oxidative stress. To correlate the long lifetime species (LLS) with lipid droplets, we performed simultaneous FLIM and two coherent nonlinear microscopy techniques: third harmonic generation (THG) imaging microscopy and coherent anti-Stokes Raman scattering (CARS) microscopy that are sensitive to lipids. We went one step further to characterize the chemical nature of this discovered species by classical Raman spectral analysis. We show application of this technique in cancer, induced pluripotent stem cell derived cardiomyocytes, as well as in freshly excised mice adipose tissue. The identified endogenous biomarker unfolds opportunities of performing non-invasive measurements of oxidative stress in vivo.
We also exploited the autofluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We performed NADH-FLIM to study the metabolic status of a vascularized three-dimensional tumor microenvironment in a microfluidic based platform. We could identify metabolically dissimilar regions, as well as identify metabolic response to anticancer drug.
Finally, we explored NADH-FLIM of a different class of organisms – bacteria. We show for the first time, FLIM-phasor fingerprint of clinically important bacteria. We discovered interesting bacterial phasor trajectories at different growth phases as well as response to antibiotics, all at single cell resolution.