Assessment of Embryo Health and Circulating Tumor Cell Metabolism Using the Phasor-FLIM Approach

Cellular functional and structural changes associated with metabolism are essential for understanding healthy tissue development and the progression of numerous diseases. Quantitatively monitoring of metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of the diseases. Unfortunately, established methods for this purpose are either destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence as a method to monitor subtle metabolic changes. In this thesis, we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic imaging in pre-implantation embryos and other biological samples.We exploited the intrinsically fluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. Using phasor-FLIM, we identified unique metabolic states that distinguish embryonic stem cells from differentiating progeny. We also apply the phasor-FLIM and hyperspectral microscopy to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological and non-invasive caliber for embryo quality. We identify the unique spectroscopic trajectories at different stages of mouse pre-implantation development which can be further used to distinguish pre-implantation embryo quality using an artificial intelligence algorithm at the early compaction stage with 86% accuracy. Furthermore, we showed the heterogeneity and changes in the normal pre-implantation embryos and aneuploidy embryos treated with the spindle assembly checkpoint inhibitor during embryo division can be rapidly distinguished at blastocyst stage via spectra phasor. Finally, we designed rapid and label-free single leukemia cell identification platform that combines high-throughput size-based separation of hemocytes, and leukemia cell identification through phasor approach and phasor-FLIM to quantify changes between free/bound NADH as an indirect measurement of metabolic alteration in living cells.These examples illustrate the potential of fluorescence lifetime imaging microscopy for unveiling complex physiological processes. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine