Two-photon Fluorescence Lifetime Imaging Microscopy Research Articles

Molecular Design of a Diketopyrrolopyrrole Conjugated Oligoelectrolyte Capable of Imaging Intracellular Vesicle Membrane Dynamics.

Conjugated oligoelectrolytes (COEs) are lipid bilayer spanning optical reporters that hold promise for delineating spatiotemporal changes in subcellular compartments. However, their ability to probe a broader range of biological processes remains restricted due to the lack of environmentally-responsive chemical functionalities. Herein, the study reports a novel COE, namely COE-KP, for monitoring spatiotemporal changes in the endolysosomal vesicles. COE-KP features a central diketopyrrolopyrrole functional group in the optically active conjugated core that confers photophysical properties suitable for bioimaging, in particular responding to the presence of hydrogen bonding functionalities within the hydrophobic domain of the lipid bilayer. COE-KP can thus discriminate cells in different growth states through two-photon fluorescence lifetime imaging microscopy (FLIM) with excitation in the NIR-II range. These changes in lifetime most reasonably reflect the degree of water permeability through the membrane and are not linked to notable differences in membrane tension or solvent polarity. Furthermore, using stimulated emission depletion (STED) nanoscopy, it is possible to directly visualize membrane-bound COEs within cellular vesicles. These results illustrate further opportunities for applying COE-based reporters for super-resolution microscopy of organelle membranes, visualization of subcellular membrane morphologies, and imaging long-term changes in membrane properties.

Measuring Metabolic Changes in Cancer Cells Using Two-Photon Fluorescence Lifetime Imaging Microscopy and Machine-Learning Analysis.

Two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) was used to track cellular metabolism with drug treatment of auto-fluorescent coenzymes NAD(P)H and FAD in living cancer cells. Simultaneous excitation at 800 nm of both coenzymes was compared with traditional sequential 740/890 nm plus another alternative of 740/800 nm, before and after adding doxorubicin in an imaging time course. Changes of redox states at single cell resolution were compared by three analysis methods: our recently introduced fluorescence lifetime redox ratio (FLIRR: NAD(P)H-a2%/FAD-a1%), machine-learning (ML) algorithms using principal component (PCA) and non-linear multi-Feature autoencoder (AE) analysis. While all three led to similar biological conclusions (early drug response), the ML models provided statistically the most robust significant results. The advantage of the single 800 nm excitation of both coenzymes for metabolic imaging in above mentioned analysis is demonstrated.

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APPSAA knock-in mouse model of Alzheimer's disease.

Reduced brain energy metabolism, mammalian target of rapamycin (mTOR) dysregulation, and extracellular amyloid beta (Aβ) oligomer (xcAβO) buildup are some well-known Alzheimer's disease (AD) features; how they promote neurodegeneration is poorly understood. We previously reported that xcAβOs inhibit nutrient-induced mitochondrial activity (NiMA) in cultured neurons. We now report NiMA disruption in vivo. Brain energy metabolism and oxygen consumption were recorded in heterozygous amyloid precursor protein knock-in (APPSAA) mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. NiMA is inhibited in APPSAA mice before other defects are detected in these Aβ-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. Glycogen synthase kinase 3 (GSK3β) signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3β with TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APPSAA mice. NiMA disruption in vivo occurs before plaques, neuroinflammation, and cognitive decline in APPSAA mice, and may represent an early stage in human AD. Amyloid beta blocks communication between lysosomes and mitochondria in vivo. Nutrient-induced mitochondrial activity (NiMA) is disrupted long before the appearance of Alzheimer's disease (AD) histopathology in heterozygous amyloid precursor protein knock-in (APPSAA/+) mice. NiMA is disrupted long before learning and memory deficits in APPSAA/+ mice. Pharmacological interventions can rescue AD-related NiMA disruption in vivo.

Simultaneous assessment of NAD(P)H and flavins with multispectral fluorescence lifetime imaging microscopy at a single excitation wavelength of 750nm.

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. The use of a multispectral detector in combination with an excitation at a single wavelength of 750nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

BackgroundHuman mesenchymal stem cells (hMSCs) utilize discrete biosynthetic pathways to self-renew and differentiate into specific cell lineages, with undifferentiated hMSCs harbouring reliance on glycolysis and hMSCs differentiating towards an osteogenic phenotype relying on oxidative phosphorylation as an energy source.MethodsIn this study, the osteogenic differentiation of hMSCs was assessed and classified over 14 days using a non-invasive live-cell imaging modality—two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This technique images and measures NADH fluorescence from which cellular metabolism is inferred.ResultsDuring osteogenesis, we observe a higher dependence on oxidative phosphorylation (OxPhos) for cellular energy, concomitant with an increased reliance on anabolic pathways. Guided by these non-invasive observations, we validated this metabolic profile using qPCR and extracellular metabolite analysis and observed a higher reliance on glutaminolysis in the earlier time points of osteogenic differentiation. Based on the results obtained, we sought to promote glutaminolysis further by using lactate, to improve the osteogenic potential of hMSCs. Higher levels of mineral deposition and osteogenic gene expression were achieved when treating hMSCs with lactate, in addition to an upregulation of lactate metabolism and transmembrane cellular lactate transporters. To further clarify the interplay between glutaminolysis and lactate metabolism in osteogenic differentiation, we blocked these pathways using BPTES and α-CHC respectively. A reduction in mineralization was found after treatment with BPTES and α-CHC, demonstrating the reliance of hMSC osteogenesis on glutaminolysis and lactate metabolism.ConclusionIn summary, we demonstrate that the osteogenic differentiation of hMSCs has a temporal metabolic profile and shift that is observed as early as day 3 of cell culture using 2P-FLIM. Furthermore, extracellular lactate is shown as an essential metabolite and metabolic fuel to ensure efficient osteogenic differentiation and as a signalling molecule to promote glutaminolysis. These findings have significant impact in the use of 2P-FLIM to discover potent approaches towards bone tissue engineering in vitro and in vivo by engaging directly with metabolite-driven osteogenesis.Graphical

Polarity-Sensitive Probe for Two-Photon Fluorescence Lifetime Imaging of Lipid Droplets In Vitro and In Vivo.

Lipid droplets (LDs) are crucial organelles used to store lipids and participate in lipid metabolism in cells. The abnormal aggregation and polarity change of LDs are associated with the occurrence of diseases, such as steatosis. Herein, the polarity-sensitive probe TBPCPP with a donor-acceptor-π-acceptor (D-A-π-A) structure was designed and synthesized. The TBPCPP has a large Stokes shift (∼220 nm), excellent photostability, high LD targeting, and considerable two-photon absorption (TPA) cross-section (∼226 GM), enabling deep two-photon imaging (∼360 μm). In addition, the fluorescence lifetime of TBPCPP decreases linearly with increasing solvent polarity. Therefore, with the assistance of two-photon fluorescence lifetime imaging microscopy (TP-FLIM), TBPCPP has successfully achieved not only the visualization of polarity changes caused by LD accumulation in HepG-2 cells but also lipid-specific imaging and visualization of different polarities in lipid-rich regions in zebrafish for the first time. Furthermore, TP-FLIM revealed that the polarity gradually decreases during steatosis in HepG-2 cells, which provided new insights into the diagnosis of steatosis.

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues.

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.

Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Water-soluble iridium(III) complexes as multicolor probes for one-photon, two-photon and fluorescence lifetime imaging

Fluorescence microscopy imaging provides an indispensable way to visualize morphological details in tissue with subcellular resolution. Of the various imaging agents currently available, heavy-metal complex based probes offer a powerful tool for live cell imaging, primarily due to their excellent photophysical properties, including evident Stokes shifts, high photoluminescence efficiency and relatively long emission lifetime of phosphorescent signals. Herein, we have designed and synthesized seven water soluble cationic iridium(III) solvato complexes (1 − 7) for live cell imaging. The emission colors of these iridium(III) complexes can be tuned from green to red in PBS buffer (phosphate buffered saline). In particular, multicolor phosphorescent imaging for cytoplasm staining of living cells has been realized. More importantly, two-photon excited imaging and fluorescence lifetime imaging microscopy have also successfully been applied for complex 7.

Single cell metabolic imaging of tumor and immune cells in vivo in melanoma bearing mice.

Metabolic reprogramming of cancer and immune cells occurs during tumorigenesis and has a significant impact on cancer progression. Unfortunately, current techniques to measure tumor and immune cell metabolism require sample destruction and/or cell isolations that remove the spatial context. Two-photon fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent metabolic coenzymes nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) provides in vivo images of cell metabolism at a single cell level. Here, we report an immunocompetent mCherry reporter mouse model for immune cells that express CD4 either during differentiation or CD4 and/or CD8 in their mature state and perform in vivo imaging of immune and cancer cells within a syngeneic B78 melanoma model. We also report an algorithm for single cell segmentation of mCherry-expressing immune cells within in vivo images. We found that immune cells within B78 tumors exhibited decreased FAD mean lifetime and an increased proportion of bound FAD compared to immune cells within spleens. Tumor infiltrating immune cell size also increased compared to immune cells from spleens. These changes are consistent with a shift towards increased activation and proliferation in tumor infiltrating immune cells compared to immune cells from spleens. Tumor infiltrating immune cells exhibited increased FAD mean lifetime and increased protein-bound FAD lifetime compared to B78 tumor cells within the same tumor. Single cell metabolic heterogeneity was observed in both immune and tumor cells in vivo. This approach can be used to monitor single cell metabolic heterogeneity in tumor cells and immune cells to study promising treatments for cancer in the native in vivo context.

Fluorescent naphthalimide boronates as theranostics: structural investigations, confocal fluorescence and multiphoton fluorescence lifetime imaging microscopy in living cells.

New design and synthetic strategies were developed to generate functional phenyl boronic acid (BA)-based fluorescent probes incorporating the 1,8-naphthalimide (NI) tag. This fluorescent core was anchored onto the BA unit through small organic linkers consisting of nitrogen groups which can arrest, and internally stabilise the phenyl-boronate units. The newly synthesised fluorophores were characterised spectroscopically by NMR spectroscopy and mass spectrometry and evaluated for their ability to bind to a naturally occurring polysaccharide, β-d-glucan in DMSO and simultaneously as act as in vitro cell imaging reagents. The uptake of these new NI-boronic acid derivatives was studied living cancer cells (HeLa, PC-3) in the presence, and absence, of β-d-glucan. Time-correlated single-photon counting (TCSPC) of DMSO solutions and two-photon fluorescence-lifetime imaging microscopy (FLIM) techniques allowed an insight into the probes' interaction with their environment. Their cellular uptake and distributions were imaged using laser scanning confocal fluorescence microscopy under single- and two-photon excitation regimes (λmax 910 nm). FLIM facilitated the estimation of the impact of the probe's cellular surroundings using the fluorophore lifetime. The extent to which this was mediated by the β-d-glucan was visualised by 2-photon FLIM in living cells. The fluorescence lifetime observed under a range of temperatures varied appreciably, indicating that changes in the environment can be sensed by these probes. In all cases, the cellular membrane penetration of these new probes was remarkable even under variable temperature conditions and localisation was widely concentrated in the cellular cytoplasm, without specific organelle trapping: we conclude that these new probes show promise for cellular imaging in living cancer cells.

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured. Both α and β cells display a wide spectrum of responses, towards either an increase or a decrease in NAD(P)H free/bound ratio. Yet, if single-cell data are averaged according to the respective donor and correlated to donor insulin secretion power, a non-random distribution of metabolic shifts emerges: robust average responses of both α and β cells towards an increase of enzyme-bound NAD(P)H belong to the donor with the lowest insulin-secretion power; by contrast, discordant responses, with α cells shifting towards an increase of free NAD(P)H and β cells towards an increase of enzyme-bound NAD(P)H, correspond to the donor with the highest insulin-secretion power. Overall, data reveal neat anti-correlation of tissue metabolic responses with respect to tissue insulin secretion power.

A FLIM photosensitizer: Targeting “Affinal” suborganelles to accelerate cancer cell oxidative stress and apoptosis

The sub-organelle targeting group is generally introduced into photosensitizer (PS) to expedite cancer cell apoptosis through oxidized specifically subcellular organelle for photodynamic therapy (PDT). Nevertheless, the exploitation of PS by targeting original and derived organelles, which might utilize a “knock-on” effect to cause rapid apoptosis, is still a huge challenge. Herein, an amphiphilic small-molecule PS, named CMOOH, was constructed by lipophilic coumarin and hydrophilic hydroxyl group, which not only anchored on the two sub-organelles with a derivational relationship (endoplasmic reticulum (ER) and lipid droplets (LDs)) but also could produce reactive oxygen species (ROS) simultaneously to facilitate the apoptosis of cancer cells under the light irradiation. Encouragingly, CMOOH firstly deciphered the apoptotic mechanism mediated by endoplasmic reticulum stress (ERS) and lipophagy by confocal laser scanning microscopy (CLSM) and two-photon fluorescence lifetime imaging microscopy (FLIM), deriving from the applicable amphipathicity, fluorescent lifetime and excellent nonlinear optical property. This tactic provided an innovative platform for treatment and imaging and renewed awareness of heightening the cancer-cell apoptosis via oxidative damage of “affinal” sub-organelles.

A Criterion of Colorectal Cancer Diagnosis Using Exosome Fluorescence-Lifetime Imaging.

This study was aimed to investigate the applicability of the exosome fluorescence-lifetime imaging microscopy (FLIM) for colorectal cancer (CRC) diagnosis. Differential ultra-centrifugation was used to extract exosomes from the blood plasma of 11 patients with colon polyps (CPs) and 13 patients with CRC at the T2-4, N0-3, and M0-1 stages. Analysis was performed using a two-photon FLIM device. In total, 165 and 195 FLIM images were recorded for the CP and CCR patient groups, respectively. Two classes of exosomes differentiated by autofluorescence average lifetime were discovered in the samples. The first class of exosomes with = (0.21 ± 0.06) ns was mostly found in samples from CRC patients. The second class with = (0.43 ± 0.19) ns was mostly found in samples from CP patients. The relative number of “CRC-associated” exosomes in the FLIM dataset was shown to be very small for the CP patient group and large for the CRC patient group. This difference was statistically significant. Therefore, the suggested CRS diagnostics criterion can be as follows. If > 0.5, the probability of CRC is high. If < 0.3, the probability of CRC is low.

Abstract 2466: Metabolic characterization of breast cancer cells and extracellular vesicles using fluorescence lifetime imaging microscopy

Abstract Extracellular vesicles (EVs) mediate cellular communication by exchanging molecules such as proteins and nucleic acids. EVs are often generated in large quantities by cancer cells and are considered a promising diagnostic biomarker that may provide information on the tumor microenvironment. Tumor cells display characteristic metabolic changes such as the Warburg effect. However, many methods to characterize the metabolism of cells or EVs are destructive or indiscriminate, losing their individual heterogeneity. There is a need to better characterize the metabolism of individual breast cancer cells and single EVs to provide insights into their heterogeneous nature and interactions in the tumor microenvironment. Two-photon fluorescence lifetime imaging microscopy (FLIM), a label-free metabolic imaging technique that can spatially map the autofluorescence properties of NAD(P)H, was used to metabolically characterize breast cancer cells and their derived EVs. EVs of size 100-1000 nm were extracted via differential ultracentrifugation from media conditioned with breast cells. Metabolic stressors including various concentrations of glucose, pyruvate, and lactate were placed on human breast cancer (MDA-MB-231) cells in culture. Subsequently, cells and EVs were imaged with FLIM to determine the metabolic relationship between individual cells and EVs. Additionally, normal human breast cells (MCF10A) and breast cancer cells (MDA-MB-231, MCF7) were imaged with FLIM to determine the metabolic signatures of the three breast cell lines. Images were analyzed using a custom EV detection algorithm, FLIM-phasor analysis, and CellPose. EV collection was validated with Nanotracker Analysis, transmission electron microscopy, and Raman spectroscopy. Human MCF10A cell populations showed significantly lower mean lifetimes than either MDA-MB-231 or MCF7 cells. This suggests that FLIM can be used to differentiate populations of cancerous and non-cancerous breast cells in an individual, non-invasive manner. MDA-MB-231 EVs showed a Gaussian and heterogenous metabolic distribution of NAD(P)H fluorescence lifetimes. This suggests a range of different NAD(P)H species that are free and protein-bound. Fluorescence lifetimes from MDA-MB-231 EVs also demonstrated a wider standard deviation from their corresponding parent cells, and a different distribution from various sub-cellular regions of the parent cell. Additionally, when metabolites available in culture media were changed to induce metabolic changes in cells, similar metabolic changes were reflected in the signatures of their released EVs. These results indicate that FLIM can be a powerful label-free non-perturbative tool to examine both single cells and EVs, as well as their heterogeneous populations, in order to further understand the concurrent metabolic properties of breast cancer cells and their released EVs. Citation Format: Elisabeth M. Martin, Janet E. Sorrells, Edita Aksamitiene, Prabuddha Mukherjee, Aneesh Alex, Eric J. Chaney, Marina Marjanovic, Stephen A. Boppart. Metabolic characterization of breast cancer cells and extracellular vesicles using fluorescence lifetime imaging microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2466.

Cancer Cell Membrane Labeling Fluorescent Doppelganger Enables In Situ Photoactivated Membrane Dynamics Tracking via Two-Photon Fluorescence Imaging Microscopy

Various suborganelles are delimited by lipid bilayers, in which high spatial and temporal morphological changes are essential to many physiological and pathological processes of cells. However, almost all the amphiphilic fluorescent molecules reported until now are not available for in situ precise tracking of membrane dynamics in cell apoptosis. Here, the MO (coumarin pyridine derivatives) was devised by engineering lipophilic coumarin and cationic pyridine salt, which not only lastingly anchored onto the plasma membrane in dark due to appropriate amphipathicity and electrostatic interactions but also in situ reflected the membrane damage and heterogeneity with secretion of extracellular vesicles (EVs) under reactive oxygen species regulation and was investigated by two-photon fluorescence lifetime imaging microscopy. This work opens up a new avenue for the development of plasma membrane staining and EV-based medicines for the early diagnosis and treatment of disease.

Probing polyvinylpyrrolidone-passivated graphene oxide nanoflakes as contrast agents inside tissue-like phantoms via multimodal confocal microscopy

Beside attractive electrical, thermal and mechanical properties, graphene oxide (GO) exhibits visible and near-infrared (NIR) photoluminescence (PL) and well-defined fingerprint Raman bands which are remarkable optical signatures to implement GO as new contrast agent for the visualization of cells or tissue, including cancer tumors. However, the biomedical use of GO as optical contrast agent is to some extent hindered by the intrinsic low emission efficiency especially at neutral pH. Herein, we successfully modulate the PL of GO nanoflakes in acidic and neutral medium by passivating them with polyvinylpyrrolidone (PVP), an amphiphilic and biocompatible polymer, thus improving the PL at pH relevant for biomedical applications. We demonstrate the potential of as-fabricated PVP-GO nanocomposites to operate as dual Raman-PL contrast agents inside tissue-like agarose-phantoms via scanning confocal Raman microscopy (CRM) under excitation at 532 nm. Super-resolution re-scan confocal microscopy (RCM) was further employed to investigate the distribution of PVP-GO inside biological phantoms at 3D level under three excitation lines (405, 488, and 561 nm). Finally, two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) at 810 nm excitation reveals the ability of PVP-GO to serve as NIR-activatable contrast agent inside tissue-like phantom. Notably, PVP coating empowers GO nanoflakes not only with enhanced optical signature, but also with excellent dispersibility inside biological phantoms, thus offering improved labeling performance of as-designed imaging contrast agent.

Structure of NAD+ consuming Acinetobacter baumannii TIR domain.

Abstract Toll-like and Interleukin-1/18 receptor (TIR) resistance proteins have been identified across biology to function as important multimodal signal transduction and immune regulatory molecules. Select TIR domain containing proteins identified in both eukaryotic and prokaryotic species have also been found to have NAD+ hydrolase activity in bacteria, plants and in mammalian cells. We report the crystal structure of the Acinetobacter baumannii Toll-Interleukin-1 receptor resistance domain protein (AbTIR) determined to a resolution of 2.8 Å, confirm its enzymatic function in NAD+ hydrolysis and map its interaction with NAD+ using HDX-MS. Additionally, we have developed a novel assay for measuring AbTIR’s enzymatic activity in live bacteria using label-free quantitative two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM). These studies provide new insight into TIR protein mechanisms and their varying roles across biology. Supported by R01 AI-082299, R21 CA191726-01

Metabolic state oscillations in cerebral nuclei detected using two-photon fluorescence lifetime imaging microscopy

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH), a key endogenous coenzyme and metabolic biomarker, can reflect the metabolic state of cells. To implement metabolic imaging of brain tissue at high resolution, we assembled a two-photon fluorescence lifetime imaging microscopy (FLIM) platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehyde-fixed mouse cerebral slices. Furthermore, NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus (SCN). The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night, and an ultradian oscillation at night was observed. Through the combination of high-resolution imaging and immunostaining data, the metabolic tendency of different cell types was detected after the first two hours of the day and at night. Thus, two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.