Search Results (208)

Background: Multiphoton tomography (MPT) is a femtosecond laser imaging technique that enables high-resolution virtual biopsies of human skin. It provides a non-invasive method for analyzing cellular metabolism, structural changes, and responses to cosmetic products, providing insights into cell–cosmetic interactions. This review explores the principles, historical development, and key applications of MPT in cosmetic research. Methods: The latest MPT device combines five modalities: (i) two-photon fluorescence: visualizes cells, elastin, and cosmetic ingredients; (ii) second harmonic generation (SHG): maps the collagen network; (iii) fluorescence lifetime imaging (FLIM): differentiates eumelanin from pheomelanin and evaluates the impact of cosmetics on cellular metabolic activity; (iv) reflectance confocal microscopy (RCM): images cell membranes and cosmetic particles; and (v) white LED imaging for dermoscopy. Results: MPT enables in-depth examination of extracellular matrix changes, cellular metabolism, and melanin production. It identifies skin responses to cosmetic products and tracks the intratissue distribution of sunscreen nanoparticles, nano- and microplastics, and other cosmetic components. Quantitative measurements, such as the elastin-to-collagen ratio, provide insights into anti-aging effects. Conclusions: MPT is a powerful in vivo imaging tool for the cosmetic industry. Its superior resolution and metabolic information facilitate the evaluation of product efficacy and support the development of personalized skincare solutions. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and its early detection can be critical for a prompt intervention that can potentially slow down the disease progression and improve the patient’s quality of life. However, a diagnosis based solely on clinical symptoms can be challenging, especially in the early stages, while the detection of specific biomarkers such as amyloid-β peptide (Aβ) and tau proteins can provide objective evidence for diagnosis. In this work, we explored the effects of Aβ peptide on cell membrane properties thanks to fluorescence lifetime imaging (FLIM) combined with the phasor analysis (FLIM-ϕ). The results showed that the membrane viscosity is altered by the presence of Aβ peptide and that cells experience this effect even at nanomolar concentrations of peptide. This considerable sensitivity opens up the possibility of envisioning a cell-based biosensor able to detect very low concentrations of Aβ in a biological fluid, thus enabling timely diagnosis and intervention. Full article

In this study, thin mouse kidney sections from healthy mice and those infected leading to acute and chronic sepsis were examined with two-photon excited fluorescence lifetime imaging (2P-FLIM) using the endogenous fluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). The results presented show that this approach is a powerful tool for investigating cell metabolism in thin tissue sections. An adapted measurement routine was established for these samples by performing a spectral scan, identifying a combination of two excitation wavelengths and two detection ranges suitable for detailed scan images of NADH and FAD. Selected positions in thin slices of the renal cortex of nine mice (three healthy, three with chronic sepsis, and three with acute sepsis) were studied using 2P-FLIM. In addition, overview images were obtained using two-photon excited fluorescence (2PEF) intensity. This study shows that healthy kidney slices differ considerably from those with acute sepsis with regard to their fluorescence lifetime signatures. The latter shows a difference in metabolism between the inner and outer cortex, indicating that outer cortical tubular cells switch their metabolism from oxidative phosphorylation to glycolysis in kidneys from mice with acute sepsis and back in later stages, as seen for mice with chronic infections. These findings suggest that 2P-FLIM could serve as a powerful tool for early-stage sepsis diagnosis and monitoring metabolic recovery during treatment. Full article

Regulation of blood glucose levels depends on the property of beta cells to couple glucose sensing with insulin secretion. This is accomplished by the concentration-dependent flux of glucose through glycolysis and oxidative phosphorylation, generating ATP. The resulting rise in cytosolic ATP/ADP inhibits K_ATP channels, inducing membrane depolarization and Ca²⁺ influx, which prompts insulin secretion. Evidence suggests that this coupling of glucose sensing with insulin secretion may be compartmentalized in the submembrane regions of the beta cell. We investigated the subcellular responses of key components involved in this coupling and found mitochondria in the submembrane zone, some tethered to the cytoskeleton near capillaries. Using Fluorescent Lifetime Imaging Microscopy (FLIM), we observed that submembrane mitochondria were the fastest to respond to glucose. In the most glucose-responsive beta cells, glucose triggers rapid, localized submembrane increases in ATP and Ca²⁺ as synchronized ~4-min oscillations, consistent with pulsatile insulin release after meals. These findings are consistent with the hypothesis that glucose sensing is coupled with insulin secretion in the submembrane zone of beta cells. This zonal adaptation would enhance both the speed and energy efficiency of beta cell responses to glucose, as only a subset of the most accessible mitochondria would be required to trigger insulin secretion. Full article

Fluorescence lifetime imaging (FLIM) has established itself as a pivotal tool for investigating biological processes within living cells. However, the extensive imaging duration necessary to accumulate sufficient photons for accurate fluorescence lifetime calculations poses a significant obstacle to achieving high-resolution monitoring of cellular dynamics. In this study, we introduce an image reconstruction method based on the edge-preserving interpolation method (EPIM), which transforms rapidly acquired low-resolution FLIM data into high-pixel images, thereby eliminating the need for extended acquisition times. Specifically, we decouple the grayscale image and the fluorescence lifetime matrix and perform an individual interpolation on each. Following the interpolation of the intensity image, we apply wavelet transformation and adjust the wavelet coefficients according to the image gradients. After the inverse transformation, the original image is obtained and subjected to noise reduction to complete the image reconstruction process. Subsequently, each pixel is pseudo-color-coded based on its intensity and lifetime, preserving both structural and temporal information. We evaluated the performance of the bicubic interpolation method and our image reconstruction approach on fluorescence microspheres and fixed-cell samples, demonstrating their effectiveness in enhancing the quality of lifetime images. By applying these techniques to live-cell imaging, we can successfully obtain high-pixel FLIM images at shortened intervals, facilitating the capture of rapid cellular events. Full article

Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α₁ after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α₁ after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening. Full article

The objectives of this study were to determine the rates of natural mortality (M), fishing mortality (F), total mortality (Z), the exploitation rates (E), as well as the biological reference points (BRPs) and the annual removal rates (R) of the crocodile shark, Pseudocarcharias kamoharai, in the Ecuadorian Pacific. Thirty similar and different models were applied to determine all these rates. These equations were obtained from studies on teleost and chondrichthyan fish. The biological parameters, including age, growth, longevity, and reproduction, were obtained from the specialized literature based on the biology of P. kamoharai in Ecuadorian waters. These biological parameters were used in all the models considered here. The M estimations were 0.14 to 0.28 based on six models for chondrichthyans and osteichthyes. These values were similar to the six algorithms designed for cartilaginous fish, ranging from 0.16 to 0.35; for this reason, these mortality rates were considered low. The Z values ranged from 0.08 to 0.51; however, they were not considered given that the three estimations were less than M, and only the Z = 0.51 was considered. Given that Z = 0.51 and M = 0.24, an F = 0.27 was obtained by subtraction, indicating a low mortality by fishing. E had values between 0.21 and 0.53, which indicated overexploitation that exceeded the E_opt = 0.50 value. The obtained BRPs were F_opt = 0.10 and 0.12 and F_lim = 0.16, which showed that the optimal fishing levels (best possible capture) to achieve long-term sustainable exploitation of the stock encompass 10 to 16% of the fishing effort applied for this species. However, the F surpassed this prudential range. The annual removal percentage (R = 21%) demonstrated that 21% of the population was being removed. Based on the biology and ecology of this species, all models applied in this study showed that P. kamoharai had low natural and fishing mortality rates and moderate total mortality; its exploitation rate exceeded the fishing limits. These values and their life history traits indicated that this shark species cannot tolerate any fishing level without threatening its populations. Full article

Understanding the deterioration processes in wooden artefacts is essential for accurately assessing their conservation status and developing effective preservation strategies. Advanced imaging techniques are currently being explored to study the impact of chemical changes on the structural and mechanical properties of wood. Nonlinear optical modalities, including second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), combined with fluorescence lifetime imaging microscopy (FLIM), offer a promising non-destructive diagnostic method for evaluating lignocellulose-based materials. In this study, we employed a nonlinear multimodal approach to examine the effects of artificially induced delignification on samples of Norway spruce (Picea abies) and European beech (Fagus sylvatica) subjected to increasing treatment durations. The integration of SHG/TPEF imaging and multi-component fluorescence lifetime analysis enabled the detection of localized variations in nonlinear signals and τ-phase of key biopolymers within wood cell walls. This methodology provides a powerful tool for early detection of wood deterioration, facilitating proactive conservation efforts of wooden artefacts. Full article

Laminopathies represent a wide range of genetic disorders caused by mutations in gene-encoding proteins of the nuclear lamina. Altered nuclear mechanics have been associated with laminopathies, given the key role of nuclear lamins as mechanosensitive proteins involved in the mechanotransduction process. To shed light on the nuclear partners cooperating with altered lamins, we focused on Src tyrosine kinase, known to phosphorylate proteins of the nuclear lamina. Here, we demonstrated a tight relationship between lamin A/C and Src in skin fibroblasts from two laminopathic patients, assessed by advanced imaging-based microscopy techniques. With confocal laser scanning and Stimulated Emission Depletion (STED) microscopy, a statistically significant higher co-distribution between the two proteins was observed in patients’ fibroblasts. Furthermore, the time-domain fluorescence lifetime imaging microscopy, combined with Förster resonance energy transfer detection, demonstrated a decreased lifetime value of Src (as donor fluorophore) in the presence of lamin A/C (as acceptor dye) in double-stained fibroblast nuclei in both healthy cells and patients’ cells, thereby indicating a molecular interaction that resulted significantly higher in laminopathic cells. All these results demonstrate a molecular interaction between Src and lamin A/C in healthy fibroblasts and their aberrant interaction in laminopathic nuclei, thus creating the possibilities of new diagnostic and therapeutic approaches for patients. Full article

The most effective method of treating tumors localized in the liver remains resection. However, in the presence of concomitant pathology, the regenerative potential of the liver is significantly reduced. To date, there is insufficient fundamental data on the mechanisms responsible for the disruption of liver regeneration, and there is no effective method for assessing its regenerative potential. The most suitable model for these purposes is acute liver injury (ALI). Modern non-contrast methods of multiphoton microscopy with second harmonic generation and fluorescence lifetime imaging microscopy (FLIM) modes enable intravital evaluation of the metabolic status of the hepatocytes; therefore, this expands the possibilities for studying the processes occurring in cells during regeneration in the context of any pathologies. Full article

The calcium cation is a crucial signaling molecule involved in numerous cellular pathways. Beyond its role as a messenger or modulator in intracellular cascades, calcium’s function in excitable cells, including nerve impulse transmission, is remarkable. The central role of calcium in nervous activity has driven the rapid development of fluorescent techniques for monitoring this cation in living cells. Specifically, genetically encoded calcium indicators (GECIs) are the most in-demand molecular tools in their class. In this work, we address two issues of calcium imaging by designing indicators based on the successful GCaMP6 backbone and the fluorescent protein BrUSLEE. The first indicator variant (GCaMP6s-BrUS), with a reduced, calcium-insensitive fluorescence lifetime, has potential in monitoring calcium dynamics with a high temporal resolution in combination with advanced microscopy techniques, such as light beads microscopy, where the fluorescence lifetime limits acquisition speed. Conversely, the second variant (GCaMP6s-BrUS-145), with a flexible, calcium-sensitive fluorescence lifetime, is relevant for static measurements, particularly for determining absolute calcium concentration values using fluorescence lifetime imaging microscopy (FLIM). To identify the structural determinants of calcium sensitivity in these indicator variants, we determine their spatial structures. A comparative structural analysis allowed the optimization of the GCaMP6s-BrUS construct, resulting in an indicator variant combining calcium-sensitive behavior in the time domain and enhanced molecular brightness. Our data may serve as a starting point for further engineering efforts towards improved GECI variants with fine-tuned fluorescence lifetimes. Full article

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 K_d < 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. Full article

In recent studies, it has been shown that fluorescence lifetime imaging (FLIM) may reveal intracellular structural details in unstained cytological preparations that are not revealed by standard staining procedures. The aim of our investigation was to examine whether FLIM images could reveal areas suggestive of polymerization in red blood cells (RBCs) of sickle cell disease (SCD) patients. We examined label-free blood films using auto-fluorescence FLIM images of 45 SCD patients and compared the results with those of 27 control persons without hematological disease. All control RBCs revealed homogeneous cytoplasm without any foci. Rounded non-sickled RBCs in SCD showed between zero and three small intensively fluorescent dots with higher lifetime values. In sickled RBCs, we found additionally larger irregularly shaped intensively fluorescent areas with increased FLIM values. These areas were interpreted as equivalent to polymerized hemoglobin. The rounded, non-sickled RBCs of SCD patients with homogeneous cytoplasm were not different from those of the erythrocytes of control patients in light microscopy. Yet, variables from the local binary pattern-transformed matrix of the FLIM values per pixel showed significant differences between non-sickled RBCs and those of control cells. In a linear discriminant analysis, using local binary pattern-transformed texture features (mean and entropy) of the erythrocyte cytoplasm of normal appearing cells, the final model could distinguish between SCD patients and control persons with an accuracy of 84.7% of the patients. When the classification was based on the examination of a single rounded erythrocyte, an accuracy of 68.5% was achieved. Employing the Linear Discriminant Analysis classifier method for machine learning, the accuracy was 68.1%. We believe that our study shows that FLIM is able to disclose the topography of the intracellular polymerization process of hemoglobin in sickle cell disease and that the images are compatible with the theory of the two-step nucleation. Furthermore, we think that the presented technique may be an interesting tool for the investigation of therapeutic inhibition of polymerization. Full article

Background: Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood. Methods: In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor. Results: Depending on the localization of the phototoxic protein, different effects on membrane microviscosity were observed. With nuclear localization of KillerRed, a gradual decrease in microviscosity was detected throughout the entire observation period, while for membrane localization of KillerRed, a dramatic increase in microviscosity was observed in the first minutes after PDT, and then a significant decrease at later stages of monitoring. The obtained data on cell monolayers are in good agreement with the data obtained for 3D tumor spheroids. Conclusions: These results indicate the involvement of membrane microviscosity in the response of tumor cells to PDT, which strongly depends on the localization of reactive oxygen species attack via targeting of a genetically encoded photosensitizer. Full article

Plant aspartic proteinases (APs) from Cynara cardunculus feature unique plant-specific insert (PSI) domains, which serve as essential vacuolar sorting determinants, mediating the transport of proteins to the vacuole. Although their role in vacuolar trafficking is well established, the exact molecular mechanisms that regulate PSI interactions and functions remain largely unknown. This study explores the ability of PSI A and PSI B to form homo- and heterodimers using a combination of pull-down assays, the mating-based split-ubiquitin system (mbSUS), and FRET-FLIM analyses. Pull-down assays provided preliminary evidence of potential PSI homo- and heterodimer formation. This was conclusively validated by the more robust in vivo mbSUS and FRET-FLIM assays, which clearly demonstrated the formation of both homo- and heterodimers between PSI A and PSI B within cellular environments. These findings suggest that PSI dimerization is related to their broader functional role, particularly in protein trafficking. Results open new avenues for future research to explore the full extent of PSI dimerization and its implications in plant cellular processes. Full article