Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach

Abstract

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or “D-trajectory”, that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.

Caffeine and Cisplatin Effectively Targets the Metabolism of a Triple-Negative Breast Cancer Cell Line Assessed via Phasor-FLIM

“…This change in signature correlates to a shift towards OXPHOS and GLY, respectively, which has been previously described as the metabolic or M-trajectory [27][28][29][30]. This trajectory has also been shown to correlate with results found in conventional biochemical assays when OXPHOS or GLY inhibitors are used to shift metabolic signatures towards one another [27,[31][32][33]. To ensure the collection of the fluorescence lifetime of NADH, all of the measurements were done using a 2-photon laser excitation set to 740 nm, and the emission was collected with an emission bandpass filter (460/40 nm) to select the emission of NADH.…”

A coarse-grained NADH redox model enables inference of subcellular metabolic fluxes from fluorescence lifetime imaging

“…Despite extensive studies and applications of FLIM in metabolic research 10–18 , it has been unclear how to relate FLIM measurements to the activities of the underlying metabolic pathways in cells. We overcome this challenge by developing a coarse-grained NADH redox model to relate FLIM measurements to mitochondrial metabolic fluxes.…”

“…Endogenous NADH has long been used to non-invasively probe cellular metabolism because NADH is autofluorescent, while NAD + is not (Heikal, 2010). Fluorescence lifetime imaging microscopy (FLIM) of NADH autofluorescence allows quantitative measurements of the concentration of NADH, the fluorescence lifetimes of NADH, and the fraction of NADH molecules bound to enzymes (Becker, 2012; Becker, 2019; Bird et al, 2005; Skala et al, 2007; Heikal, 2010; Sharick et al, 2018; Sanchez et al, 2018; Sanchez et al, 2019; Ma et al, 2019). It has been observed that the fraction of enzyme-bound NADH and NADH fluorescence lifetimes are correlated with the activity of oxidative phosphorylation, indicating that there is a connection between NADH enzyme-binding and mitochondrial metabolic fluxes (Bird et al, 2005; Skala et al, 2007).…”

“…Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo 10–18 . However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria.…”

A Perspective on Expanding Our Understanding of Cancer Treatments by Integrating Approaches from the Biological and Physical Sciences

“…This method of imaging has been adapted for bulk measurements by utilizing a plate reader 63 or for distinct objects by coupling it with a microscope. 64,65 By expanding this setup to multiphoton excitation on a microscope, phototoxicity of the sample is decreased and deeper penetration is possible for large tissue samples of up to 3 mm. 66 This technique can also be coupled with confocal imaging methods to measure cell morphological parameters or fluorescent markers of cell death.…”