An official website of the United States government
Abstract A major challenge in ART is to select high-quality oocytes and embryos. The metabolism of oocytes and embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. Here, we review recent work on noninvasive metabolic imaging of cumulus cells, oocytes, and embryos. We focus our discussion on fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent coenzymes NAD(P)H and flavine adenine dinucleotide (FAD+), which play central roles in many metabolic pathways. FLIM measurements provide quantitative information on NAD(P)H and FAD+ concentrations and engagement with enzymes, leading to a robust means of characterizing the metabolic state of cells. We argue that FLIM is a promising approach to aid in oocyte and embryo selection.
more »
« less
Label-Free Optical Metabolic Imaging in Cells and Tissues
Over the last half century, the autofluorescence of the metabolic cofactors NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) has been quantified in a variety of cell types and disease states. With the spread of nonlinear optical microscopy techniques in biomedical research, NADH and FAD imaging has offered an attractive solution to noninvasively monitor cell and tissue status and elucidate dynamic changes in cell or tissue metabolism. Various tools and methods to measure the temporal, spectral, and spatial properties of NADH and FAD autofluorescence have been developed. Specifically, an optical redox ratio of cofactor fluorescence intensities and NADH fluorescence lifetime parameters have been used in numerous applications, but significant work remains to mature this technology for understanding dynamic changes in metabolism. This article describes the current understanding of our optical sensitivity to different metabolic pathways and highlights current challenges in the field. Recent progress in addressing these challenges and acquiring more quantitative information in faster and more metabolically relevant formats is also discussed. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 25 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
more »
« less
- Award ID(s):
- 1846853
- PAR ID:
- 10417419
- Date Published:
- Journal Name:
- Annual Review of Biomedical Engineering
- Volume:
- 25
- Issue:
- 1
- ISSN:
- 1523-9829
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract BackgroundMacrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. MethodsHere, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. ResultsBased on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. ConclusionsOverall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.more » « less
-
Nicotinamide adenine dinucleotide (NADH) is a cofactor that serves to shuttle electrons during metabolic processes such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS). NADH is autofluorescent, and its fluorescence lifetime can be used to infer metabolic dynamics in living cells. Fiber-coupled time-correlated single photon counting (TCSPC) equipped with an implantable needle probe can be used to measure NADH lifetime in vivo, enabling investigation of changing metabolic demand during muscle contraction or tissue regeneration. This study illustrates a proof of concept for point-based, minimally-invasive NADH fluorescence lifetime measurement in vivo. Volumetric muscle loss (VML) injuries were created in the left tibialis anterior (TA) muscle of male Sprague Dawley rats. NADH lifetime measurements were collected before, during, and after a 30[Formula: see text]s tetanic contraction in the injured and uninjured TA muscles, which was subsequently fit to a biexponential decay model to yield a metric of NADH utilization (cytoplasmic vs protein-bound NADH, the A[Formula: see text]/A[Formula: see text] ratio). On average, this ratio was higher during and after contraction in uninjured muscle compared to muscle at rest, suggesting higher levels of free NADH in contracting and recovering muscle, indicating increased rates of glycolysis. In injured muscle, this ratio was higher than uninjured muscle overall but decreased over time, which is consistent with current knowledge of inflammatory response to injury, suggesting tissue regeneration has occurred. These data suggest that fiber-coupled TCSPC has the potential to measure changes in NADH binding in vivo in a minimally invasive manner that requires further investigation.more » « less
-
The extracellular matrix (ECM) is a dynamic and complex microenvironment that modulates cell behavior and cell fate. Changes in ECM composition and architecture have been correlated with development, differentiation, and disease progression in various pathologies, including breast cancer [1]. Studies have shown that aligned fibers drive a pro-metastatic microenvironment, promoting the transformation of mammary epithelial cells into invasive ductal carcinomaviathe epithelial-to-mesenchymal transition (EMT) [2]. The impact of ECM orientation on breast cancer metabolism, however, is largely unknown. Here, we employ two non-invasive imaging techniques, fluorescence-lifetime imaging microscopy (FLIM) and intensity-based multiphoton microscopy, to assess the metabolic states of cancer cells cultured on ECM-mimicking nanofibers in a random and aligned orientation. By tracking the changes in the intrinsic fluorescence of nicotinamide adenine dinucleotide and flavin adenine dinucleotide, as well as expression levels of metastatic markers, we reveal how ECM fiber orientation alters cancer metabolism and EMT progression. Our study indicates that aligned cellular microenvironments play a key role in promoting metastatic phenotypes of breast cancer as evidenced by a more glycolytic metabolic signature on nanofiber scaffolds of aligned orientation compared to scaffolds of random orientation. This finding is particularly relevant for subsets of breast cancer marked by high levels of collagen remodeling (e.g. pregnancy associated breast cancer), and may serve as a platform for predicting clinical outcomes within these subsets [3–6].more » « less
-
null (Ed.)Abstract We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1146/annurev-bioeng-071516-044730
