This study employs the optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of human neutrophils in vitro, under a variety of activation conditions and targeted metabolic pathway inhibition at the single-cell level. By measuring effector functions such as oxidative burst, neutrophil activation could be confirmed. In addition, identical metabolic changes were detected in zebrafish larvae neutrophils following activation, confirming the validity of these findings in vivo.
Metabolic features of mitosis remain poorly understood because this phase of the cell cycle is rapid and heterogeneous between cells within a dish. Label-free optical metabolic imaging (OMI) can monitor rapid changes in cell metabolism with single cell resolution using two-photon microscopy of the optical redox ratio (NAD(P)H/FAD) and NAD(P)H fluorescence lifetimes. Here, we brought together image analysis tools to quantify OMI time-courses of single cells undergoing mitosis across multiple cell lines. Alignment of OMI and morphological features over time provided unique insight into metabolic changes during mitosis within unperturbed systems.
SignificanceAdvancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging).AimThis perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs.ApproachThree key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended.ConclusionImprovements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.
Significance: Fluorescence lifetime imaging microscopy (FLIM) measures the decay rate of fluorophores, thus providing insights into molecular interactions. FLIM is a powerful molecular imaging technique that is widely used in biology and medicine.
Aim: This perspective highlights some of the major advances in FLIM instrumentation, analysis, and biological and clinical applications that we have found impactful over the last year.
Approach: Innovations in FLIM instrumentation resulted in faster acquisition speeds, rapid imaging over large fields of view, and integration with complementary modalities such as single-molecule microscopy or light-sheet microscopy. There were significant developments in FLIM analysis with machine learning approaches to enhance processing speeds, fit-free techniques to analyze images without a priori knowledge, and open-source analysis resources. The advantages and limitations of these recent instrumentation and analysis techniques are summarized. Finally, applications of FLIM in the last year include label-free imaging in biology, ophthalmology, and intraoperative imaging, FLIM of new fluorescent probes, and lifetime-based Förster resonance energy transfer measurements.
Conclusions: A large number of high-quality publications over the last year signifies the growing interest in FLIM and ensures continued technological improvements and expanding applications in biomedical research.
KEYWORDS: Fluorescence lifetime imaging, Luminescence, Microscopy, In vivo imaging, Modulation, Fluorescence resonance energy transfer, Tumors, Image segmentation, Molecules, Biological research
Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.
Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.
Approach: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed.
Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.
Significance: Glioblastoma multiforme (GBM) is the most frequently diagnosed adult primary brain malignancy with poor patient prognosis. GBM can recur despite aggressive treatment due to therapeutically resistant glioblastoma stem cells (GSCs) that may exhibit metabolic plasticity.
Aim: Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence can be acquired with fluorescence lifetime imaging microscopy (FLIM) to examine its bound and free metabolic states in GSC and GBM tissues.
Approach: We compared the mean NADH fluorescence lifetime in live human GSCs and normal neural stem cells and validated those results by measuring oxygen consumption rates (OCRs). We also examined the role that invasive versus less-invasive GSCs had on tumor metabolism by measuring the mean NADH lifetimes and the relative amount of the longer-lived component of NADH and correlated these results with survival in an orthotopic mouse xenograft model.
Results: Mean NADH lifetime, amount of bound NADH, and OCR were increased in GSCs. Compared with normal mouse brain, mean NADH lifetimes were longer for all GBM tissues. Invasive xenografts had higher relative amounts of the longer-lived NADH component, and this correlated with decreased survival.
Conclusions: FLIM offers cellular resolution quantification of metabolic flux in GBM phenotypes, potentially informing biomedical researchers on improved therapeutic approaches.
The cell cycle is extensively characterized, yet there is much to learn about the decision-making process involved in cell division. Here, the optical redox ratio (ratio of NADH to FAD fluorescence intensity) of MCF10A cells was imaged every 20 minutes over 12 hours using multiphoton microscopy. Cell tracking was used to monitor individual cells over time. We found a positive correlation in the variations of the optical redox ratio with the phases of the entire cell cycle. This study reveals the novel role of redox signaling in the progression the cell cycle.
Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.
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