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Nonmuscle myosin II (NMII) generates cytoskeletal forces that drive cell division, embryogenesis, muscle contraction and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here, we describe a Förster resonance energy transfer (FRET)-based tension sensor that can detect myosin-associated force along the filamentous actin network. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMII isoform B (NMIIB) exhibit significant spatial and temporal heterogeneity as a function of donor lifetime and fluorophore energy exchange. These measurements provide a proxy for inferred forces that vary widely along the actin cytoskeleton. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aβ) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.

[Display omitted] Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid–liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process. [ABSTRACT FROM AUTHOR]

Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be associated with clinical subtypes of alpha-synucleinopathies, like Parkinson's disease and multiple system atrophy. However, direct evidence is lacking due to the caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in vitro method to explore various alpha-synuclein (αsyn) aggregate morphotypes. We performed a spectroscopic investigation of the HEK293 cell model, transfected with human wildtype-αsyn and A53T-αsyn variants, using the amyloid fibril-specific heptameric luminescent oligomeric thiophene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant Kd < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The ensuing intracellular aggregates were stained with h-FTAA followed by an evaluation of the spectral features and fluorescence lifetime of intracellular αsyn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA, based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in vitro and in vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early subtype-specific diagnosis is of the utmost importance for prognosis and treatment response. The potential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtype-specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation-specific features of various protein pathologies. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging microscopy is sensitive to molecular interactions and environments. In homo-dyne frequency-domain fluorescence lifetime imaging microscopy, images of fluorescence objects are acquired at different phase settings of the detector. The detected intensity as a function of detector phase is a sinusoidal function that is sensitive to the lifetime of the fluorescent species. In this paper, the theory of phase-sensitive fluorescence image correlation spectroscopy is described. In this version of lifetime imaging, image correlation spectroscopy analysis (i.e., spatial autocorrelation) is applied to successive fluorescence images acquired at different phase settings of the detector. Simulations of different types of lifetime distributions reveal that the phase-dependent density of fluorescent objects is dependent on the heterogeneity of lifetimes present in the objects. We provide an example of this analysis workflow to a cervical cancer cell stained with a fluorescent membrane probe. [ABSTRACT FROM AUTHOR]

Islet transplantation represents a promising therapeutic approach for diabetes management, yet the isolation and evaluation of pancreatic islets remain challenging. This study focuses on the isolation of islets from rabbit pancreases, followed by a comprehensive assessment of their viability and functionality. We developed a novel method for isolating islet cells from the pancreas of adult rabbits. We successfully isolated viable islets, which were subsequently evaluated through a combination of viability assays, an insulin enzyme-linked immunosorbent assay (ELISA), and fluorescence lifetime imaging microscopy (FLIM). The viability assays indicated a high percentage of intact islets post-isolation, while the insulin ELISA demonstrated robust insulin secretion in response to glucose stimulation. FLIM provided insights into the metabolic state of the islets, revealing distinct fluorescence lifetime signatures correlating with functional viability. Our findings underscore the potential of rabbit islets as a model for studying islet biology and diabetes therapy, highlighting the efficacy of combining traditional assays with advanced imaging techniques for comprehensive functional assessments. This research contributes to the optimization of islet isolation protocols and enhances our understanding of islet functional activity dynamics in preclinical settings. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment‐specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age‐dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment‐dependent manner during ageing. [ABSTRACT FROM AUTHOR]

Objective: A number of previously synthesized fluorogenic arylidene-imidazolones, which predominantly stain the endoplasmic reticulum (ER) of living cells, were studied by time-resolved fluorescence microscopy. It was suggested that the use of fluorescence microscopy of this type can enhance the selectivity of ER staining. Methods: The lifetimes of arylidene-imidazolone compounds in set of solvents with different polarity were measured with time-correlated single photon counting spectroscopy. Live HeLa Kyoto cells were stained with studied dyes and analyzed with time-resolved fluorescence microscopy. Results and discussion: It was found that most of the studied compounds show bi- or triexponential fluorescence decay patterns in solutions. In live cell culture dye (I) showed a monoexponential decay pattern, while dyes (II–IV) were better fitted by a biexponential function. Dyes (I), (III), and (IV) stained both ER and adiposomes, while dye (II) stained only ER. Conclusions: It is shown that under FLIM conditions discriminative filtering of cellular organelles stained with studied fluorogenic dyes is possible and applicable if the difference of mean amplitude-weighted lifetime is more than 0.1 ns, thus increasing the selectivity of ER staining in live cells. [ABSTRACT FROM AUTHOR]

Mitochondria are commonly recognized as the powerhouses of the cell, primarily responsible for energy production through oxidative phosphorylation. Alongside this vital function, they also play crucial roles in regulating calcium signaling, maintaining membrane potential, and modulating apoptosis. Their involvement in various cellular pathways becomes particularly evident during oogenesis and embryogenesis, where mitochondrial quantity, morphology, and distribution are tightly controlled. The efficiency of the mitochondrial network is maintained through multiple quality control mechanisms that are essential for reproductive success. These include mitochondrial unfolded protein response, mitochondrial dynamics, and mitophagy. Not surprisingly, mitochondrial dysfunction has been implicated in infertility and ovarian aging, prompting investigation into mitochondria as diagnostic and therapeutic targets in assisted reproduction. To date, mitochondrial DNA copy number in oocytes, cumulus cells, and trophectoderm biopsies, and fluorescent lifetime imaging microscopy-based assessment of NADH and flavin adenine dinucleotide content have been explored as potential predictors of embryo competence, yielding limited success. Despite challenges in the clinical application of mitochondrial diagnostic strategies, these enigmatic organelles have a significant impact on reproduction, and their potential role as diagnostic targets in assisted reproduction is likely to remain an active area of investigation in the foreseeable future. [ABSTRACT FROM AUTHOR]

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with

Tumor cells invade normal surrounding tissues through continuous division. In this study, we hypothesized that cell cycle regulation changes the immune efficacy of ovarian cancer. To investigate this hypothesis, a Förster resonance energy transfer (FRET) sensor was constructed to characterize the cell activity in real time. Cell shrinkage caused by apoptosis induces the aggregation of proteins on the cell membrane, leading to variations in the fluorescence lifetime of FRET sensors. Moreover, we tracked cell activity across various cycles following co-culture with an immune checkpoint inhibitor. Consequently, we assessed how cell cycle regulation influences immunotherapy in a tumor mouse model. This approach, which involves inhibiting typical cell cycle processes, markedly enhances the effectiveness of immunotherapy. Our findings suggest that modulating the cycle progression of cancer cells may represent a promising approach to enhance the immune response of ovarian cancer cells and the efficacy of immunotherapy based on immune checkpoint inhibitors. [ABSTRACT FROM AUTHOR]

The use of fluorescence in imaging has been a pivotal factor in advancing our scientific understanding. As microscopy continues to evolve, the terminology used to describe these techniques becomes increasingly complex, often resulting in a bewildering array of acronyms that resemble alphabet soup. Among the most prominent acronyms are those associated with advanced fluorescence microscopy techniques: Fluorescence Loss in Photobleaching (FLIP), Fluorescence Localization after Photobleaching (FLAP), Fluorescence Recovery after Photobleaching (FRAP), Förster Resonance Energy Transfer (FRET), and Fluorescence Lifetime Imaging Microscopy (FLIM). Each of these methods provides invaluable insights into molecular dynamics and interactions within living cells and tissues. This article, Part 2 of the Alphabet Soup of Microscopy series, aims to clarify these widely used fluorescence microscopy techniques and to illuminate their contributions to our understanding of cellular processes. [ABSTRACT FROM AUTHOR]

Microalgae are the richest source of natural carotenoids—accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis—gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging. [ABSTRACT FROM AUTHOR]

Summary: Woody plants display some photosynthetic activity in stems, but the biological role of stem photosynthesis and the specific contributions of bark and wood to carbon uptake and oxygen evolution remain poorly understood.We aimed to elucidate the functional characteristics of chloroplasts in stems of different ages in Fraxinus ornus. Our investigation employed diverse experimental approaches, including microsensor technology to assess oxygen production rates in whole stem, bark, and wood separately. Additionally, we utilized fluorescence lifetime imaging microscopy (FLIM) to characterize the relative abundance of photosystems I and II (PSI : PSII chlorophyll ratio) in bark and wood.Our findings revealed light‐induced increases in O2 production in whole stem, bark, and wood. We present the radial profile of O2 production in F. ornus stems, demonstrating the capability of stem chloroplasts to perform light‐dependent electron transport. Younger stems exhibited higher light‐induced O2 production and dark respiration rates than older ones.While bark emerged as the primary contributor to net O2 production under light conditions, our data underscored that wood chloroplasts are also photosynthetically active. The FLIM analysis unveiled a lower PSI abundance in wood than in bark, suggesting stem chloroplasts are not only active but also acclimate to the spectral composition of light reaching inner compartments. [ABSTRACT FROM AUTHOR]

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling. [ABSTRACT FROM AUTHOR]

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)‐based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1‐containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM‐FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.

Purpose: This study is aiming to test whether variation in post warming culture time impacts blastocyst metabolism or pregnancy outcome. Methods: In this single center retrospective cohort study, outcomes of 11,520 single frozen embryo transfer (FET) cycles were analyzed from January 2015 to December 2020. Patient treatments included both natural and programmed cycles. Time categories were determined using the time between blastocyst warming and embryo transfer: 0 (0- <1h), 1 (1-<2h), 2 (2-<3h), 3(3-<4h), 4 (4-<5), 5 (5-<6), 6 (6-<7) and 7 (7-8h). Non-invasive metabolic imaging of discarded human blastocysts for up to 10h was also performed using Fluorescence lifetime imaging microscopy (FLIM) to examine for metabolic perturbations during culture. Results: The mean age of patients across all time categories were comparable (35.6 ± 3.9). Live birth rates (38-52%) and miscarriage rate (5-11%) were not statistically different across post-warming culture time. When assessing pregnancy outcomes based on the use of PGT-A, miscarriage and live birth rates were not statistically different across culture hours in both PGT-A and non-PGT cycles. Further metabolic analysis of blastocysts for the duration of 10h of culture post warming, revealed minimal metabolic changes of embryos in culture. Conclusion: Overall, our results show that differences in the time of post warming culture have no significant impact on miscarriage or live birth rate for frozen embryo transfers. This information can be beneficial for clinical practices with either minimal staffing or a high number of patient cases. [ABSTRACT FROM AUTHOR]

Genetically encoded fluorescent biosensors provide an attractive means of measuring chemical changes in single cells on fast timescales (milliseconds to seconds). While their most prominent application has been in tracking neural activity and neurotransmitter release, there has been growing interest in developing and deploying new versions of these tools to study brain metabolism. However, the careful use of these tools and the interpretation of the data they provide remain challenging. Many biosensors are subject to interferences that can alter sensor responses within a single cell or between cells, producing ambiguous results. This presents a challenge for quantitation and for our ability to accurately interpret sensor responses. This review describes current methods of sensor quantitation, with a focus on cellular interferences that commonly affect sensor performance, ways to avoid false inferences, and recent advances in sensor optimization to make them more robust. [ABSTRACT FROM AUTHOR]

The ongoing expansion of fifth-generation (5G) and future sixth-generation (6G) mobile communications is expected to result in widespread human exposure to millimeter-wave (mmWave) radiation globally. Given the short penetration depth of mmWaves and their high absorption by the skin, it is imperative to investigate the potential effects of 5G radiation not only in terms of temperature increase but also at the cellular level. To understand the biological mechanisms of mmWave effects, accurate methods for assessing mmWave absorption in the skin are crucial. In this study, we use fluorescence lifetime imaging microscopy (FLIM) to explore these effects. Employing a mmWave exposure system operating at 26 gigahertz (GHz), porcine skin is irradiated for varying durations (5, 10, 20, and 30 min). We investigate changes in tissue temperature and the autofluorescence of flavin adenine dinucleotide (FAD). Our findings suggest that operating our mmWave exposure systems at the configured power level of 26 GHz is unlikely to cause damage to FADs, even after a 30 min exposure duration. [ABSTRACT FROM AUTHOR]

Omega-3 polyunsaturated fatty acids (PUFA) found primarily in fish oil have been a popular supplement for cardiovascular health because they can substantially reduce circulating triglyceride levels in the bloodstream to prevent atherosclerosis. Beyond this established extracellular activity, here, we report a mode of action of PUFA, regulating intracellular triglyceride metabolism and lipid droplet (LD) dynamics. Real-time imaging of the subtle and highly dynamic changes of intracellular lipid metabolism was enabled by a fluorescence lifetime probe that addressed the limitations of intensity-based fluorescence quantifications. Surprisingly, we found that among omega-3 PUFA, only docosahexaenoic acid (DHA) promoted the lipolysis in LDs and reduced the overall fat content by approximately 50%, and consequently helped suppress macrophage differentiation into foam cells, one of the early steps responsible for atherosclerosis. Eicosapentaenoic acid, another omega-3 FA in fish oil, however, counteracted the beneficial effects of DHA on lipolysis promotion and cell foaming prevention. These in vitro findings warrant future validation in vivo. [ABSTRACT FROM AUTHOR]

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)‐based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1‐containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM‐FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells. [ABSTRACT FROM AUTHOR]

Background: Microvessels in tumor tissue play a crucial role in meeting the metabolic needs of transformed cells, controlling the entry of xenobiotics into tumor tissue, and regulating local inflammation that promotes metastasis. Methylene blue has photosensitizing properties and can also affect dysfunctional mitochondria. Methods: The study was performed on the primary culture of CECs. The cells underwent photodynamic treatment through 660 nm laser irradiation at a power density of 300 mW/cm2. The MTT, TMRE, and TUNEL assays were used to assess the survival, redox metabolism, mitochondrial activity, and apoptosis of CECs. Additionally, the metabolic activity of cells was evaluated using FLIM by measuring the fluorescence lifetime of NADH and FAD. Results: When CECs were incubated with MB, there was an increase in mitochondrial activity that was dependent on the concentration of MB. Additionally, mitochondrial activity increased when the CECs were exposed to 660 nm laser irradiation at an energy dose of up to 5 J/cm2. Following PDT, a slight shift towards oxidative phosphorylation was observed. Conclusions: In vitro application of MB accumulation or laser irradiation causes a shift in the redox status of CECs towards increased reducing activity, without causing any cell damage. However, the combined action of PS and laser radiation has the opposite effect on the redox status of cells, resulting in an increase in the oxidized form of FAD. [ABSTRACT FROM AUTHOR]

The successful trajectory of liposome-encapsulated doxorubicin (e.g., Doxil, which has been approved by the U.S. Food and Drug Administration) as an anticancer nanodrug in clinical applications is contradicted by in vitro cell viability data that highlight its reduced efficacy in promoting cell death compared with non-encapsulated doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated doxorubicin (L-DOX) in several in vitro cellular models. Cell entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.

Abstract The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Fluorescence lifetime imaging microscopy (FLIM) is a technique that analyzes the metabolic state of tissues based on the spatial distribution of fluorescence lifetimes of certain interacting molecules. We used multiphoton FLIM to study the metabolic state of developing C57BL6/J and rd10 retinas based on the fluorescence lifetimes of free versus bound nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H), with free NAD(P)H percentages suggesting increased glycolysis and bound NAD(P)H percentages indicating oxidative phosphorylation. The mice were sacrificed and enucleated at various time points throughout their first 3 months of life. The isolated eyecups were fixed, sectioned using a polyacrylamide gel embedding technique, and then analyzed with FLIM. The results suggested that in both C57BL6/J mice and rd10 mice, oxidative phosphorylation initially decreased and then increased, plateauing over time. This trend, however, was accelerated in rd10 mice, with its turning point occurring at p10 versus the p30 turning point in C57BL6/J mice. There was also a noticeable difference in oxidative phosphorylation rates between the outer and inner retinas in both strains, with greater oxidative phosphorylation present in the latter. A greater understanding of rd10 and WT metabolic changes during retinal development may provide deeper insights into retinal degeneration and facilitate the development of future treatments.

Background: Coenzyme Q10 (CoQ10), also known as ubiquinone-10, is an important molecule of the mitochondrial respiratory chain that acts as an electron carrier between complexes I, II, and III and additionally functions as an antioxidant. Due to its bioenergetic properties, CoQ10 is of high interest for therapeutic and cosmetic use. This study aims to characterize the metabolic impact of CoQ10 on primary human dermal fibroblasts (HDF) using fluorescence lifetime imaging microscopy (FLIM) and electron microscopy. Methods: FLIM of nicotinamide adenine dinucleotide (NADH) is a robust method to characterize cellular energy metabolism that also provides spatial information. Electron microscopy offers a way to characterize the ultrastructure of mitochondria and reveal features not visible in FLIM. Results: We reported a shift towards longer lifetimes of NADH in primary fibroblasts from ten different donors upon treatment with CoQ10, which indicates the stimulation of oxidative phosphorylation. This is confirmed by phasor-based metabolic pattern segmentation, which showed localization of longer NADH lifetimes in CoQ10-treated cells, indicating activated mitochondria in the cytoplasm. In addition, a complementary investigation of the mitochondrial ultrastructure using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography showed a reduction in stress granules in CoQ10-treated cells. Conclusions: Together, FLIM and electron microscopy (EM) imaging strongly imply that CoQ10 stimulates cellular energy metabolism.

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism.

Artificial aggregation‐induced emission luminogens (AIEgens) have flourished in bio‐applications with the development of synthetic chemistry, which however are plagued by issues like singularity in structures and non‐renewability. The unique structures and renewability of biomass moieties can compensate for these drawbacks, but their properties are hard to design and regulate due to their confined structures. Therefore, it appears to be a reasonable approach to derive AIEgens from abundant biomass (BioAIEgens), integrating the bilateral advantages of both synthetic and natural AIEgens. In this work, the blue‐violet emissive coumarin with its lactone structure serving as a rare natural acceptor, is utilized to construct donor‐π‐acceptor typed BioAIE isomers incorporating the propeller‐like and electron‐donating triphenylamine (TPA) unit. The results show that Cm‐p‐TPA undergoes charge transfer with its keto form, emitting red light at 600 nm, which can be applied to monitor Cu2+ concentration during mitophagy using fluorescence lifetime imaging microscopy because of the excellent biocompatibility, photostability, and specific recognition to Cu2+. This work not only demonstrates the feasibility of utilizing positional isomerization to modulate excited‐state evolutions and resultant optical properties, but also provides evidence for the rationality of constructing biologically‐active BioAIEgens via a biomass‐derivatization concept. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Does fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging assessment of human blastocysts prior to frozen transfer correlate with pregnancy outcomes? SUMMARY ANSWER FLIM failed to distinguish consistent patterns in mitochondrial metabolism between blastocysts leading to pregnancy compared to those that did not. WHAT IS KNOWN ALREADY FLIM measurements provide quantitative information on NAD(P)H and flavin adenine dinucleotide (FAD+) concentrations. The metabolism of embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. STUDY DESIGN, SIZE, DURATION This was a pilot trial enrolling 121 IVF couples who consented to have their frozen blastocyst measured using non-invasive metabolic imaging. After being warmed, 105 couples' good-quality blastocysts underwent a 6-min scan in a controlled temperature and gas environment. FLIM-assessed blastocysts were then transferred without any intervention in management. PARTICIPANTS/MATERIALS, SETTING, METHODS Eight metabolic parameters were obtained from each blastocyst (4 for NAD(P)H and 4 for FAD): short and long fluorescence lifetime, fluorescence intensity, and fraction of the molecule engaged with enzyme. The redox ratio (intensity of NAD(P)H)/(intensity of FAD) was also calculated. FLIM data were combined with known metadata and analyzed to quantify the ability of metabolic imaging to differentiate embryos that resulted in pregnancy from embryos that did not. De-identified discarded aneuploid human embryos (n = 158) were also measured to quantify correlations with ploidy status and other factors. Statistical comparisons were performed using logistic regression and receiver operating characteristic (ROC) curves with 5-fold cross-validation averaged over 100 repeats with random sampling. AUC values were used to quantify the ability to distinguish between classes. MAIN RESULTS AND THE ROLE OF CHANCE No metabolic imaging parameters showed significant differences between good-quality blastocysts resulting in pregnancy versus those that did not. A logistic regression using metabolic data and metadata produced an ROC AUC of 0.58. In contrast, robust AUCs were obtained when classifying other factors such as comparison of Day 5 (n = 64) versus Day 6 (n = 41) blastocysts (AUC = 0.78), inner cell mass versus trophectoderm (n = 105: AUC = 0.88) and aneuploid (n = 158) versus euploid and positive pregnancy embryos (n = 108) (AUC = 0.82). LIMITATIONS, REASONS FOR CAUTION The study protocol did not select which embryo to transfer and the cohort of 105 included blastocysts were all high quality. The study was also limited in number of participants and study sites. Increased power and performing the trial in more sites may have provided a stronger conclusion regarding the merits of the use of FLIM clinically. WIDER IMPLICATIONS OF THE FINDINGS FLIM failed to distinguish consistent patterns in mitochondrial metabolism between good-quality blastocysts leading to pregnancy compared to those that did not. Blastocyst ploidy status was, however, highly distinguishable. In addition, embryo regions and embryo day were consistently revealed by FLIM. While metabolic imaging detects mitochondrial metabolic features in human blastocysts, this pilot trial indicates it does not have the potential to serve as an effective embryo viability detection tool. This may be because mitochondrial metabolism plays an alternative role post-implantation. STUDY FUNDING/COMPETING INTEREST(S) This study was sponsored by Optiva Fertility, Inc. Boston IVF contributed to the clinical site and services. Becker Hickl, GmbH, provided the FLIM system on loan. T.S. was the founder and held stock in Optiva Fertility, Inc. and D.S. and E.S. had options with Optiva Fertility, Inc. during this study. TRIAL REGISTRATION NUMBER The study was approved by WCG Connexus IRB (Study Number 1298156). [ABSTRACT FROM AUTHOR]

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings. [ABSTRACT FROM AUTHOR]

Response of lipid bilayers to external mechanical stimuli is an active area of research with implications for fundamental and synthetic cell biology. Developing novel tools for systematically imposing mechanical strains and non-invasively mapping out interfacial (membrane) stress distributions on lipid bilayers can accelerate research in this field. We report a miniature platform to manipulate model cell membranes in the form of droplet interface bilayers (DIBs), and non-invasively measure spatio-temporally resolved interfacial stresses using two photon fluorescence lifetime imaging of an interfacially active molecular flipper (Flipper-TR). We established the effectiveness of the developed framework by investigating interfacial stresses accompanying three key processes associated with DIBs: thin film drainage between lipid monolayer coated droplets, bilayer formation, and bilayer separation. The measurements revealed fundamental aspects of DIBs including the existence of a radially decaying interfacial stress distribution post bilayer formation, and the simultaneous build up and decay of stress respectively at the bilayer corner and center during bilayer separation. Finally, utilizing interfacial rheology measurements and MD simulations, we also reveal that the tested molecular flipper is sensitive to membrane fluidity that changes with interfacial stress - expanding the scientific understanding of how molecular flippers sense stress. [ABSTRACT FROM AUTHOR]

This article discusses the development of a novel pH probe called Mem-C0C18 for quantitative monitoring of cell surface pH using fluorescence lifetime imaging. The surface pH of tumor cells is lower than that of normal cells due to the production of acidic metabolites. The probe targets the plasma membrane and accurately measures pH changes using fluorescence lifetime imaging. The article provides details on the molecular design of the probe, its spectral properties, establishment of standard curves, and its application in evaluating the effects of extracellular fluid acidity and glycolysis on cell surface pH. The probe shows promise as a tool for studying pH fluctuations in the cell membrane. [Extracted from the article]

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior. [ABSTRACT FROM AUTHOR]

Fibroblasts are among the most common cell types in the stroma responsible for creating and maintaining the structural organization of the extracellular matrix in the dermis, skin regeneration, and a range of immune responses. Until now, the processes of fibroblast adaptation and functioning in a varying environment have not been fully understood. Modern laser microscopes are capable of studying fibroblasts in vitro and ex vivo. One‐photon‐ and two‐photon‐excited fluorescence microscopy, Raman spectroscopy/microspectroscopy are well‐suited noninvasive optical methods for fibroblast imaging in vitro and ex vivo. In vivo staining‐free fibroblast imaging is not still implemented. The exception is fibroblast imaging in tattooed skin. Although in vivo noninvasive staining‐free imaging of fibroblasts in the skin has not yet been implemented, it is expected in the future. This review summarizes the state‐of‐the‐art in fibroblast visualization using optical methods and discusses the advantages, limitations, and prospects for future noninvasive imaging. [ABSTRACT FROM AUTHOR]

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aβ) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.

Nanotechnology research offers advancements in materials and industrial uses such as nano-electronics, medical sciences, fuel, biology, and technological innovation. The most often employed nanomaterial is Graphene oxide (GO), which has been regarded as a dominant progenitor for many specialized industries. The current work aims to establish a new, cost-effective, easy, and straightforward GO synthesis method. Laser spectroscopy and even traditional tools have been utilized for characterization. The prepared GO has been confirmed by XRD and FT-IR spectroscopy. Laser spectroscopy based on confocal microscopes like Raman micro-spectroscopy and fluorescence lifetime imaging microscopy (FLIM) has been used to ensure the synthesis of GO. Also, it could help in the detection of the prepared layer thickness. Raman spectroscopy detects the number of GO layers, which is one on average, while the Raman mapping shows a good distribution of GO on the substrate. Based on excitation with a 445 nm pulsed laser, FLIM shows that mono GO layers cover most of the surface while only a tiny area contains more than a single layer. The novel aspect of this work is the method of preparation and the way of utilizing both FLIM and Raman to detect the number of GO layers. The prepared GO could be used in many applications like solar cells and filtration.

Abstract Background Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1. Methods We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed. Results EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca2+ concentration. Conclusions Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report direct blind unmixing of lifetimes with subnanosecond resolution and subpixel spatial resolution using standard raster scan FLIM images. We demonstrate our method using a wide range of simulated and experimental data.

The biological relevance of nitric oxide (NO) and reactive oxygen species (ROS) in signaling, metabolic regulation, and disease treatment has become abundantly clear. The dramatic change in NO/ROS processing that accompanies a changing oxygen landscape calls for new imaging tools that can provide cellular details about both [O2 ] and the production of reactive species. Myoglobin oxidation to the met state by NO/ROS is a known sensor with absorbance changes in the visible range. We previously employed Förster resonance energy transfer to read out the deoxygenation/oxygenation of myoglobin, creating the subcellular [O2 ] sensor Myoglobin-mCherry. We now add the fluorescent protein EYFP to this sensor to create a novel probe that senses both met formation, a proxy for ROS/NO exposure, and [O2 ]. Since both proteins are present in the construct, it can also relieve users from the need to measure fluorescence lifetime, making [O2 ] sensing available to a wider group of laboratories.

RELEVANCE A special place in the development of enteral insufficiency is given to dysproteinemia, which is one of the leading causes of the development of decubital ulcers in patients with spinal cord injury. Early enteral nutrition partially solved this problem, but the incidence of bedsores still remains high and reaches 68%. The risk of metabolic disorders in the acute period of spinal injury is largely determined by non-occlusive intestinal ischemia against the background of spinal shock, neurohumoral dysregulation; intra-intestinal and intra-abdominal hypertension; change in intestinal microflora. Pathological changes in the intestinal wall occur during the first 20 days after injury and further exacerbate chronic maldigestion, malabsorption, intestinal dyskinesia in patients with traumatic spinal cord disease. New knowledge about the features of early enteral nutrition in patients in the acute period of traumatic spinal cord disease will reduce the risk of decubitus ulcerative defects.AIM OF THE STUDY To study the dynamics of metabolic processes in the tissues of the small intestine in the acute period of spinal injury.MATERIAL AND METHODS Wistar rats (n=22). Spinal injury was simulated by acute complete transection of the spinal cord at the level of Th5–Th6 vertebrae. The assessment of metabolic changes in the cells of the serous membrane of the intestine was performed immediately, 3 and 24 hours after injury. The metabolism was assessed in vivo using fluorescence time-resolved macroimaging technology FLIM by autofluorescence in the spectral channel of the metabolic cofactor nicotinamide adenine dinucleotide (phosphate).RESULTS The acute period of spinal cord injury is accompanied by a change in the endogenous autofluorescence of the serous membrane of the small intestine: a statistically significant decrease in the mean fluorescence lifetime (τm), the lifetime of the long component (τ2), and the relative contribution of the long component (а2) in 24 h after injury was recorded. The changes observed using FLIM confirm the catabolic type of metabolism in the tissues of the small intestine after spinal cord injury.CONCLUSION For the first time in the experiment in vivo it has been shown that the acute period of spinal injury is accompanied by a violation of metabolic processes in the tissues of the small intestine. This fact requires a more balanced approach in calculating the calorie content of nutrients used for early enteral nutrition in patients with spinal cord injury.

Abstract Artificial aggregation‐induced emission luminogens (AIEgens) have flourished in bio‐applications with the development of synthetic chemistry, which however are plagued by issues like singularity in structures and non‐renewability. The unique structures and renewability of biomass moieties can compensate for these drawbacks, but their properties are hard to design and regulate due to their confined structures. Therefore, it appears to be a reasonable approach to derive AIEgens from abundant biomass (BioAIEgens), integrating the bilateral advantages of both synthetic and natural AIEgens. In this work, the blue‐violet emissive coumarin with its lactone structure serving as a rare natural acceptor, is utilized to construct donor‐π‐acceptor typed BioAIE isomers incorporating the propeller‐like and electron‐donating triphenylamine (TPA) unit. The results show that Cm‐p‐TPA undergoes charge transfer with its keto form, emitting red light at 600 nm, which can be applied to monitor Cu2+ concentration during mitophagy using fluorescence lifetime imaging microscopy because of the excellent biocompatibility, photostability, and specific recognition to Cu2+. This work not only demonstrates the feasibility of utilizing positional isomerization to modulate excited‐state evolutions and resultant optical properties, but also provides evidence for the rationality of constructing biologically‐active BioAIEgens via a biomass‐derivatization concept.

Treatment of various diseases, in particular cancer, usually requires the targeting of biologically active molecules at a selected subcellular compartment. We modified our previously developed modular nanotransporters (MNTs) for targeting mitochondria. The new MNTs are capable of binding to the protein predominantly localized on the outer mitochondrial membrane, Keap1. These MNTs possessing antiKeap1 monobody co-localize with mitochondria upon addition to the cells. They efficiently interact with Keap1 both in solution and within living cells. A conjugate of the MNT with a photosensitizer, chlorin e6, demonstrated significantly higher photocytotoxicity than chlorin e6 alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles. [ABSTRACT FROM AUTHOR]

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1. Methods: We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed. Results: EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca2+ concentration. Conclusions: Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology. [ABSTRACT FROM AUTHOR]

The viscosity and crowding of biological environment are considered vital for the correct cellular function, and alterations in these parameters are known to underly a number of pathologies including diabetes, malaria, cancer and neurodegenerative diseases, to name a few. Over the last decades, fluorescent molecular probes termed molecular rotors proved extremely useful for exploring viscosity, crowding, and underlying molecular interactions in biologically relevant settings. In this review, we will discuss the basic principles underpinning the functionality of these probes and will review advances in their use as sensors for lipid order, protein crowding and conformation, temperature and non‐canonical nucleic acid structures in live cells and other relevant biological settings. [ABSTRACT FROM AUTHOR]

Genetically encoded fluorescence lifetime biosensors have emerged as powerful tools for quantitative imaging, enabling precise measurement of cellular metabolites, molecular interactions, and dynamic cellular processes. This review provides an overview of the principles, applications, and advancements in quantitative imaging with genetically encoded fluorescence lifetime biosensors using fluorescence lifetime imaging microscopy (go-FLIM). We highlighted the distinct advantages of fluorescence lifetime-based measurements, including independence from expression levels, excitation power, and focus drift, resulting in robust and reliable measurements compared to intensity-based approaches. Specifically, we focus on two types of go-FLIM, namely Förster resonance energy transfer (FRET)–FLIM and single-fluorescent protein (FP)-based FLIM biosensors, and discuss their unique characteristics and benefits. This review serves as a valuable resource for researchers interested in leveraging fluorescence lifetime imaging to study molecular interactions and cellular metabolism with high precision and accuracy. [ABSTRACT FROM AUTHOR]