scholarly journals Metabolic state of human blastocysts measured by fluorescence lifetime imaging microscopy

2021 ◽  
Author(s):  
Marta Venturas ◽  
Jaimin S Shah ◽  
Xingbo Yang ◽  
Tim H Sanchez ◽  
William Conway ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Cell Mass ◽  
Reproductive Technologies ◽  
Metabolic Imaging ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Embryo Viability ◽  
Metabolic State ◽  
Lifetime Imaging ◽  
Non Invasive

Mammalian embryos undergo large changes in metabolism over the course of preimplantation development. Embryo metabolism has long been linked to embryo viability, suggesting its potential utility in Assisted Reproductive Technologies (ART) to aid in selecting high quality embryos. However, the metabolism of human embryos remains poorly characterized due to a lack of non-invasive methods to measure their metabolic state. Here, we explore the application of metabolic imaging via fluorescence lifetime imaging microscopy (FLIM) for studying human blastocysts. We use FLIM to measure the autofluorescence of two central coenzymes, NAD(P)H and FAD+, in 215 discarded human blastocysts from 137 patients. We find that FLIM is sensitive enough to detect significant metabolic differences between blastocysts. We show that the metabolic state of human blastocysts changes continually over time, and that variations between blastocyst are partially explained by both the time since fertilization and their developmental stage, but not their morphological grade. We also observe significant metabolic heterogeneity within individual blastocysts, including between the inner cell mass and the trophectoderm, and between the portions of hatching blastocysts within and without the zona pellucida. Taken together, this work reveals novel aspects of the metabolism of human blastocysts and suggests that FLIM is a promising approach to assess embryo viability through non-invasive, quantitative measurements of their metabolism.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ning Ma ◽  
Nabora Reyes de Mochel ◽  
Paula Duyen Pham ◽  
Tae Yeon Yoo ◽  
Ken W. Y. Cho ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Embryo Quality ◽  
Assisted Reproductive Technologies ◽  
Learning Algorithm ◽  
Reproductive Technologies ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Embryo Viability ◽  
Experimental Conditions ◽  
Lifetime Imaging

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.

scholarly journals Metabolic Imaging Using Two-Photon Excited NADH Intensity and Fluorescence Lifetime Imaging

2012 ◽  
Vol 18 (4) ◽  
pp. 761-770 ◽  
Author(s):  
Jorge Vergen ◽  
Clifford Hecht ◽  
Lyandysha V. Zholudeva ◽  
Meg M. Marquardt ◽  
Richard Hallworth ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Organ Of Corti ◽  
Metabolic Imaging ◽  
Fluorescence Lifetime Imaging ◽  
Metabolic State ◽  
Intact Mouse ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Reduced Nicotinamide Adenine Dinucleotide

AbstractMetabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

scholarly journals Raman Imaging and Fluorescence Lifetime Imaging Microscopy for Diagnosis of Cancer State and Metabolic Monitoring

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5682
Author(s):  
Lucas Becker ◽  
Nicole Janssen ◽  
Shannon L. Layland ◽  
Thomas E. Mürdter ◽  
Anne T. Nies ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Fluorescence Lifetime ◽  
Drug Efficacy ◽  
Raman Microspectroscopy ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Non Invasive

Hurdles for effective tumor therapy are delayed detection and limited effectiveness of systemic drug therapies by patient-specific multidrug resistance. Non-invasive bioimaging tools such as fluorescence lifetime imaging microscopy (FLIM) and Raman-microspectroscopy have evolved over the last decade, providing the potential to be translated into clinics for early-stage disease detection, in vitro drug screening, and drug efficacy studies in personalized medicine. Accessing tissue- and cell-specific spectral signatures, Raman microspectroscopy has emerged as a diagnostic tool to identify precancerous lesions, cancer stages, or cell malignancy. In vivo Raman measurements have been enabled by recent technological advances in Raman endoscopy and signal-enhancing setups such as coherent anti-stokes Raman spectroscopy or surface-enhanced Raman spectroscopy. FLIM enables in situ investigations of metabolic processes such as glycolysis, oxidative stress, or mitochondrial activity by using the autofluorescence of co-enzymes NADH and FAD, which are associated with intrinsic proteins as a direct measure of tumor metabolism, cell death stages and drug efficacy. The combination of non-invasive and molecular-sensitive in situ techniques and advanced 3D tumor models such as patient-derived organoids or microtumors allows the recapitulation of tumor physiology and metabolism in vitro and facilitates the screening for patient-individualized drug treatment options.

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