Endogenous autofluorescence of biological tissues is an important source of information for biomedical diagnostics. Despite the molecular complexity of biological tissues, the list of commonly known fluorophores is strictly limited. Still, the question of molecular sources of the red and near-infrared excited autofluorescence remains open. In this work we demonstrated that the oxidation products of organic components (lipids, proteins, amino acids, etc.) can serve as the molecular source of such red and near-infrared excited autofluorescence. Using model solutions and cell systems (human keratinocytes) under oxidative stress induced by UV irradiation we demonstrated that oxidation products can contribute significantly to the autofluorescence signal of biological systems in the entire visible range of the spectrum, even at the emission and excitation wavelengths higher than 650 nm. The obtained results suggest the principal possibility to explain the red fluorescence excitation in a large class of biosystems—aggregates of proteins and peptides, cells and tissues—by the impact of oxidation products, since oxidation products are inevitably presented in the tissue. The observed fluorescence signal with broad excitation originated from oxidation products may also lead to the alteration of metabolic imaging results and has to be taken into account.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIββ3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets’ aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells’ aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

Edema, i.e., fluid accumulation in the interstitial space, accompanies numerous pathological states of the human organism, including heart failure (HF), inflammatory response, and lymphedema. Nevertheless, techniques for quantitative assessment of the edema’s severity and dynamics are absent in clinical practice, and the analysis is mainly limited to physical examination. This fact stimulates the development of novel methods for fast and reliable diagnostics of fluid retention in tissues. In this work, we focused on the possibilities of two microscopic techniques, nailfold video capillaroscopy (NVC) and confocal laser scanning microscopy (CLSM), in the assessment of the short-term and long-term cutaneous edema. We showed that for the patients with HF, morphological parameters obtained by NVC—namely, the apical diameter of capillaries and the size of the perivascular zone—indicate long-term edema. On the other hand, for healthy volunteers, the application of two models of short-term edema, venous occlusion, and histamine treatment of the skin, did not reveal notable changes in the capillary parameters. However, a significant reduction of the NVC image sharpness was observed in this case, which was suggested to be due to water accumulation in the epidermis. To verify these findings, we made use of CLSM, which provides the skin structure with cellular resolution. It was observed that for the histamine-treated skin, the areas of the dermal papillae become hyporefractive, leading to the loss of contrast and the lower visibility of capillaries. Similar effect was observed for patients undergoing infusion therapy. Collectively, our results reveal the parameters can be used for pericapillary edema assessment using the NVC and CLSM, and paves the way for their application in a clinical set-up.

Anaemia, a decrease in total concentration of haemoglobin (Hb) in blood, affects substantial percentage of the population worldwide. Currently, the gold standard for determining the Hb level is the invasive analysis of venous blood. Yet, more and more research groups demonstrate the possibility of non-invasive Hb assessment using white light imaging of tissue sites where Hb is the main chromophore, in particular, fingernails. Despite the promising declarations, non-invasive Hb assessment via RGB-imaging is still poorly used in practice. The main reason is the difficulty in establishing the true accuracy of the methods presented in different works since they are tested on private datasets collected under different experimental conditions. Here we present an open dataset containing RGB images of skin and fingernails for patients with a known level of Hb, thus providing a single benchmark for researchers and engineers in the field, aimed at fostering translation of non-invasive imaging methods to the bedside.

SignificanceAutofluorescence 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.AimWe 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.ApproachWe 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.ResultsThe use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm 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.ConclusionsThe approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm 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.

Non-invasive methods for determining blood hemoglobin (Hb) concentration are urgently needed to avoid the painful and time-consuming process of invasive venous blood sampling. Many such methods rely on assessing the average attenuation of light over a tissue area where hemoglobin is the dominant chromophore, without separating those areas corresponding to vessels and bloodless tissue. In this study, we investigate whether it is possible to determine hemoglobin levels in the blood by assessing the changes in light intensity when passing through large vessels in comparison to adjacent tissues, using this as a Hb level predictor. Using Monte Carlo light transport modeling, we evaluate the accuracy of determining hemoglobin levels via light intensity contrast and vessel widths estimated in the transmittance illumination geometry and estimate the influence of physiologically significant parameters such as vessel depth, dermis vascularization, and melanin content in the epidermis on the blood Hb prediction error. The results show that physiological variations in tissue parameters limit the mean absolute error of this method to ~15 g/L for blood Hb levels varying in the 60–160 g/L range, which finding is also supported by experimental data obtained for volunteers with different total blood Hb levels that have been determined invasively. We believe the application of new approaches to the non-invasive assessment of Hb levels will lead to the creation of reliable and accurate devices that are applicable in point-of-care and clinical practice.