Fluorescence Lifetime Imaging Research Articles

Single-sample image-fusion upsampling of fluorescence lifetime images.

Fluorescence lifetime imaging microscopy (FLIM) provides detailed information about molecular interactions and biological processes. A major bottleneck for FLIM is image resolution at high acquisition speeds due to the engineering and signal-processing limitations of time-resolved imaging technology. Here, we present single-sample image-fusion upsampling, a data-fusion approach to computational FLIM super-resolution that combines measurements from a low-resolution time-resolved detector (that measures photon arrival time) and a high-resolution camera (that measures intensity only). To solve this otherwise ill-posed inverse retrieval problem, we introduce statistically informed priors that encode local and global correlations between the two "single-sample" measurements. This bypasses the risk of out-of-distribution hallucination as in traditional data-driven approaches and delivers enhanced images compared, for example, to standard bilinear interpolation. The general approach laid out by single-sample image-fusion upsampling can be applied to other image super-resolution problems where two different datasets are available.

Fluorophore multimerization on a PEG backbone as a concept for signal amplification and lifetime modulation

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels’ fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Unlocking the potential of different types of biomass‐derived carbon dots as fluorescence lifetime imaging probes

The ongoing search for innovative and environmentally friendly luminescent materials coupled with customizable physical and chemical properties at the nanoscale positions carbon dots (CDots) as ideal candidates for photonic applications. However, even today, their rational design for specific applications remains elusive due to the unknown relationship between optical and structural properties. Therefore, this study aims to fill this gap by linking the chemical structure of the precursor and synthetic conditions to the final structural composition of the CDots and, by extension, to their optical properties. The study shows that while CDots are chemically stable, their optical properties, which are determined by the carbonaceous core and surface groups, are highly pH dependent. These properties, together with the long fluorescence lifetimes observed in living cells (&gt; 10 ns), make these biomass‐derived CDots promising probes for time‐resolved fluorescence imaging.

Multimodal Multiphoton Tomography with a Compact Femtosecond Fiber Laser

Multiphoton tomography (MPT) based on near infrared (NIR) femtosecond laser technology has become a versatile high-resolution clinical and research imaging tool. We report on multimodal MPT with the air-cooled fiber laser tomograph MPTcompact. The ultracompact passively mode-locked erbium-doped 50/80 MHz laser operating at 780 nm is directly integrated into the 360° imaging head mounted on a flexible mechanical arm. The tomograph provides optical biopsies with subcellular resolution and optical metabolic imaging capability based on two-photon autofluorescence (AF), second harmonic generation, fluorescence lifetime imaging (FLIM) by time-correlated single photon counting with 250 ps temporal resolution, reflectance confocal microscopy, and white LED light CMOS camera imaging. For the first time, NIR femtosecond laser pulses have been used on human skin to realize simultaneous one-photon confocal imaging and two-photon imaging. Most useful information is provided by the two-photon excited intratissue AF of the coenzymes NAD(P)H and flavins. The signals of both types of coenzymes can be separated by FLIM. Furthermore, the free and protein-bound forms can be distinguished by time-resolved AF detection because the protein-bound NADH has one order higher AF lifetime than free (non-bound) NADH. The tomograph contains onboard storage batteries so that it can operate for up to a few hours independently from external power supply. Applications include in vivo cancer detection and in situ evaluation of anti-aging drugs and pharmaceuticals. The tomograph MPTcompact has been successfully tested in a clinical multicenter study for the diagnosis of malignant melanoma on 100 patients with suspicious pigmented lesions. MPT has the potential to realize non-invasive high-resolution label-free in vivo histology within minutes and, therefore, to reduce the number of physically taken biopsies.

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy.

To demonstrate the first near-infrared adaptive optics fluorescence lifetime imaging ophthalmoscopy (NIR-AOFLIO) measurements in vivo of the human retinal pigment epithelial (RPE) cellular mosaic and to visualize lifetime changes at different retinal eccentricities. NIR reflectance and autofluorescence were captured using a custom adaptive optics scanning light ophthalmoscope in 10 healthy subjects (23-64 years old) at seven eccentricities and in two eyes with retinal abnormalities. Repeatability was assessed across two visits up to 8weeks apart. Endogenous retinal fluorophores and hydrophobic whole retinal extracts of Abca4-/- pigmented and albino mice were imaged to probe the fluorescence origin of NIR-AOFLIO. The RPE mosaic was resolved at all locations in five of seven younger subjects (<35 years old). The mean lifetime across near-peripheral regions (8° and 12°) was longer compared to near-foveal regions (0° and 2°). Repeatability across two visits showed moderate to excellent correlation (intraclass correlation: 0.88 [τm], 0.75 [τ1], 0.65 [τ2], 0.98 [a1]). The mean lifetime across drusen-containing eyes was longer than in age-matched healthy eyes. Fluorescence was observed in only the extracts from pigmented Abca4-/- mouse. NIR-AOFLIO was repeatable and allowed visualization of the RPE cellular mosaic. An observed signal in only the pigmented mouse extract infers the fluorescence signal originates predominantly from melanin. Variations observed across the retina with intermediate age-related macular degeneration suggest NIR-AOFLIO may act as a functional measure of a biomarker for in vivo monitoring of early alterations in retinal health.

Surfactant-Dependent Partitioning of Organics in Aqueous-Organic Reaction Systems.

A fluorescence lifetime imaging microscopy (FLIM) technique characterizes surfactant-dependent partitioning of organics in a system that mimics a Negishi-like cross-coupling reaction in water, under synthetic concentrations, with emulsion droplets. Experimental partitioning data were not predictable from simple hydrophilic-lipophilic balances. The ionic surfactant cetrimonium chloride suppressed the reactivity of the metallic zinc surface, presumably through competitive chloride binding and concurrent cetrimonium coating, a finding that may contribute to the reduced performance of ionic surfactants in the bench-scale coupling reaction.

Fluorescence saturation imaging microscopy: molecular fingerprinting with a standard confocal microscope.

Molecular specificity in fluorescence imaging of cells and tissues can be increased by measuring parameters other than intensity. For instance, fluorescence lifetime imaging became a widespread modality for biomedical optics. Previously, we suggested using the fluorescence saturation effect at pulsed laser excitation to map the absorption cross-section as an additional molecular contrast in two-photon microscopy [Opt. Lett.47(17), 4455 (2022).10.1364/OL.465605]. Here, it is shown that, somewhat counterintuitive, fluorescence saturation can be observed under cw excitation in a standard confocal microscopy setup. Mapping the fluorescence saturation parameter allows obtaining additional information about the fluorophores in the system, as demonstrated by the example of peptide hydrogel, stained cells and unstained thyroid gland. The suggested technique does not require additional equipment and can be implemented on confocal systems as is.

Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in Arabidopsis.

Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the Arabidopsis PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. In vitro and in vivo, PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the pdi9 mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the ire1a-ire1b mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.

Chemical Control of Fluorescence Lifetime towards Multiplexing Imaging.

Fluorescence lifetime imaging has been a powerful tool for biomedical research. Recently, fluorescence lifetime-based multiplexing imaging has expanded imaging channels by using probes that harbor the same spectral channels and distinct excited state lifetime. While it is desirable to control the excited state lifetime of any given fluorescent probes, the rational control of fluorescence lifetimes remains a challenge. Herein, we chose boron dipyrromethene (BODIPY) as a model system and provided chemical strategies to regulate the fluorescence lifetime of its derivatives with varying spectral features. We find electronegativity of structural substituents at the 8' and 5' positions is important to control the lifetime for the green-emitting and red-emitting BODIPY scaffolds. Mechanistically, such influences are exerted via the photo-induced electron transfer and the intramolecular charge transfer processes for the 8' and 5' positions of BODIPY, respectively. Based on these principles, we have generated a group of BODIPY probes that enable imaging experiments to separate multiple targets using fluorescence lifetime as a signal. In addition to BODIPY, we envision modulation of electronegativity of chemical substituents could serve as a feasible strategy to achieve rational control of fluorescence lifetime for a variety of small molecule fluorophores.

Label-free metabolic optical biomarkers track stem cell fate transition in real time.

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective.

Recognition of multi-component compounds based on occurrence time of secondary crest in the fluorescence lifetime attenuation curve

Fluorescence lifetime is the main characteristic parameter of fluorescence. It is a widely used to draw fluorescence lifetime attenuation curves and to fit fluorescence lifetime parameters by using gated detection methods to identify the species of substances. However, the fluorescence attenuation of each fluorophore in a multi-component compound interferes with one another, affecting the accuracy of identification. In this paper, we propose a method to accurately identify substances by using the occurrence time of the secondary crest of the fluorescence lifetime attenuation curve based on the principle of gated detection to measure the fluorescence lifetime. Furthermore, we design a fluorescence lifetime imaging measurement system and select the same areas of interest in the images for analysis and comparison. The average lifetime of the fluorescence and the occurrence time of the secondary crest are considered as the characteristic parameters. We use five commercially available motor engine oils as the experimental samples and compare the recognition performance of different kernel functions based on a support vector machine (SVM). The radial basis kernel function presents the best performance in terms of recognition accuracy and speed. The recognition rates of the SVM model with the average fluorescence lifetime and the occurrence time of the secondary crest in the attenuation curve of the fluorescence lifetime as a feature vector are 76.24% and 74.65%, respectively. The recognition rate of the SVM model which combines them as feature vectors reaches 91.88%. The experimental results demonstrate that the occurrence time of the secondary crest in the attenuation curve of the fluorescence lifetime can be employed as the basis for substance identification in the analysis of the fluorescence characteristics of multi-component compounds, whose recognition accuracy is similar to the average fluorescence lifetime parameter. Moreover, the occurrence time of the secondary crest of the fluorescence lifetime attenuation curve can be implemented to identify multi-component compounds when it is used as a characteristic parameter.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Metabolic imaging of human cumulus cells reveals associations with pregnancy and live birth.

Can fluorescence lifetime imaging microscopy (FLIM) detect associations between the metabolic state of cumulus cell (CC) samples and the clinical outcome of the corresponding embryos? FLIM can detect significant variations in the metabolism of CC associated with the corresponding embryos that resulted in a clinical pregnancy versus those that did not. CC and oocyte metabolic cooperativity are known to be necessary for the acquisition of developmental competence. However, reliable CC biomarkers that reflect oocyte viability and embryo developmental competency have yet to be established. Quantitative measures of CC metabolism could be used to aid in the evaluation of oocyte and embryo quality in ART. A prospective observational study was carried out. In total, 223 patients undergoing IVF with either conventional insemination or ICSI at a tertiary care center from February 2018 to May 2020 were included, with no exclusion criteria applied. This cohort had a mean maternal age of 36.5 ± 4.4 years and an average oocyte yield of 16.9 (range 1-50). One to four CC clusters from each patient were collected after oocyte retrieval and vitrified. CC metabolic state was assessed using FLIM to measure the autofluorescence of the molecules NAD(P)H and FAD+, which are essential for multiple metabolic pathways. CC clusters were tracked with their corresponding oocytes and associated embryos. Patient age, Day 3 and Day 5/6 embryo morphological grades, and clinical outcomes of embryos with traceable fate were recorded. Nine FLIM quantitative parameters were obtained for each CC cluster. We investigated associations between the FLIM parameters and patient maternal age, embryo morphological rank, ploidy, and clinical outcome, where false discovery rate P-values of <0.05 were considered statistically significant. A total of 851 CC clusters from 851 cumulus-oocyte complexes from 223 patients were collected. Of these CC clusters, 623 were imaged using FLIM. None of the measured CC FLIM parameters were correlated with Day 3 morphological rank or ploidy of the corresponding embryos, but FAD+ FLIM parameters were significantly associated with morphological rank of blastocysts. There were significant differences for FAD+ FLIM parameters (FAD+ fraction engaged and short lifetime) from CC clusters linked with embryos resulting in a clinical pregnancy compared with those that did not, as well as for CC clusters associated with embryos that resulted in a live birth compared those that did not. Our data are based on a relatively low number of traceable embryos from an older patient population. Additionally, we only assessed CCs from 1 to 4 oocytes from each patient. Future work in a younger patient population with a larger number of traceable embryos, as well as measuring the metabolic state of CCs from all oocytes from each patient, would provide a better understanding of the potential utility of this technology for oocyte/embryo selection. Metabolic imaging via FLIM is able to detect CC metabolic associations with maternal age and detects variations in the metabolism of CCs associated with oocytes leading to embryos that result in a clinical pregnancy and a live birth versus those that do not. Our findings suggest that FLIM of CCs may be used as a new approach to aid in the assessment of oocyte and embryo developmental competence in clinical ART. National Institutes of Health grant NIH R01HD092550-03 (to C.R., and D.J.N.). Becker and Hickl GmbH and Boston Electronics sponsored research with the loaning of equipment for FLIM. D.J.N. and C.R. are inventors on patent US20170039415A1. N/A.

Layer-controllable synthesis, lattice structural Determination, and fluorescence lifetime imaging of (HA)2(MA)n-1PbnI3n+1 Quasi-2D organic-inorganic perovskite microsheets

Hybrid quasi-two-dimensional (quasi-2D) perovskites have attracted great interest due to their excellent photovoltaic and light-emitting properties, which enable the rapid development of high-performance perovskite-based solar cells, light-emitting diodes, and lasers. Although many efforts have been made to improve the quality and stability of organic-inorganic perovskites, the controlled synthesis of high-quality layered perovskites remains challenging. Here, single-crystalline quasi-2D (HA)2(MA)n-1PbnI3n+1 perovskites with layer numbers of n from 1 to 3 have been synthesized in a controllable manner, exhibiting stable lattice structures and tunable optical emission. Structurally, hexylamine (HA) with longer alkyl chains was used as an organic spacer in Ruddlesden-Popper (RP) phase perovskites to improve the chemical stability of perovskites. Various ratios of precursor materials were adopted for separating pure and large-scale quasi-2D (HA)2(MA)n-1PbnI3n+1 perovskites, in which different layer-number perovskites with tunable band gaps were obtained with the modulated fluorescence lifetimes. Our lattice structural and fluorescence-lifetime characterizations of RP-phase perovskites provide critical information on the emerging quasi-2D perovskite-crystal systems and their fluorescence relaxation dynamics. The employed strategy to synthesize high-quality perovskites (HA)2(MA)n-1PbnI3n+1 is technically essential to develop the rising perovskite-based photovoltaic and light-emitting applications.

Pioglitazone Phases and Metabolic Effects in Nanoparticle-Treated Cells Analyzed via Rapid Visualization of FLIM Images.

Fluorescence lifetime imaging microscopy (FLIM) has proven to be a useful method for analyzing various aspects of material science and biology, like the supramolecular organization of (slightly) fluorescent compounds or the metabolic activity in non-labeled cells; in particular, FLIM phasor analysis (phasor-FLIM) has the potential for an intuitive representation of complex fluorescence decays and therefore of the analyzed properties. Here we present and make available tools to fully exploit this potential, in particular by coding via hue, saturation, and intensity the phasor positions and their weights both in the phasor plot and in the microscope image. We apply these tools to analyze FLIM data acquired via two-photon microscopy to visualize: (i) different phases of the drug pioglitazone (PGZ) in solutions and/or crystals, (ii) the position in the phasor plot of non-labelled poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), and (iii) the effect of PGZ or PGZ-containing NPs on the metabolism of insulinoma (INS-1 E) model cells. PGZ is recognized for its efficacy in addressing insulin resistance and hyperglycemia in type 2 diabetes mellitus, and polymeric nanoparticles offer versatile platforms for drug delivery due to their biocompatibility and controlled release kinetics. This study lays the foundation for a better understanding via phasor-FLIM of the organization and effects of drugs, in particular, PGZ, within NPs, aiming at better control of encapsulation and pharmacokinetics, and potentially at novel anti-diabetics theragnostic nanotools.

Net O2 exchange rates under dark and light conditions across different stem compartments.

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.

Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

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.

Fluorescence lifetime imaging microscopy of flexible and rigid dyes probes the biophysical properties of synthetic and biological membranes

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C12, the flipper dye FliptR, as well as the dyes 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C16, lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and Bodipy C12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic dyes, molecular rotors, or flipper in a wide range of biophysics experiments. Compared to other methods, fluorescence lifetime imaging is a minimally invasive and nondestructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis, and the probe’s intracellular distribution, as well as potential side effects induced by labeling and imaging.

In Vitro Effect of 9,9'-Norharmane Dimer against Herpes Simplex Viruses.

Herpes simplex virus (HSV) infections are highly widespread among humans, producing symptoms ranging from ulcerative lesions to severe diseases such as blindness and life-threatening encephalitis. At present, there are no vaccines available, and some existing antiviral treatments can be ineffective or lead to adverse effects. As a result, there is a need for new anti-HSV drugs. In this report, the in vitro anti-HSV effect of 9,9'-norharmane dimer (nHo-dimer), which belongs to the β-carboline (βC) alkaloid family, was evaluated. The dimer exhibited no virucidal properties and did not impede either the attachment or penetration steps of viral particles. The antiviral effect was only exerted under the constant presence of the dimer in the incubation media, and the mechanism of action was found to involve later events of virus infection. Analysis of fluorescence lifetime imaging data showed that the nHo-dimer internalized well into the cells when present in the extracellular incubation medium, with a preferential accumulation into perinuclear organelles including mitochondria. After washing the host cells with fresh medium free of nHo-dimer, the signal decreased, suggesting the partial release of the compound from the cells. This agrees with the observation that the antiviral effect is solely manifested when the alkaloid is consistently present in the incubation media.