Multifarious analytical capabilities of the UV/Vis protein fluorescence in blood plasma.

Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for ∼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

In vivo sensing of cutaneous edema: A comparative study of diffuse reflectance, Raman spectroscopy and multispectral imaging.

Quantitative noninvasive assessment of water content in tissues is important for biomedicine. Optical spectroscopy is potentially capable of solving this problem; however, its applicability for clinical diagnostics remains questionable. The presented study compares diffuse reflectance spectroscopy, Raman spectroscopy and multispectral imaging in the characterization of cutaneous edema. The source-detector geometries for each method are selected based on Monte Carlo simulations results to detect the signal from the dermis. Then, the kinetics of the edema development is studied for two models. All methods demonstrate synchronous trends for histamine-induced edema: The water content reaches a maximum of 1 hour after histamine application and then gradually decreases. For the venous occlusion, a 51% increase in water content is observed with Raman spectroscopy. The differences in water content estimation by three methods are explained based on the light propagation model. The obtained results are essential for introducing quantitative optical water measurement technology to the clinics.

Single-Fiber Diffuse Reflectance Spectroscopy and Spatial Frequency Domain Imaging in Surgery Guidance: A Study on Optical Phantoms.

Diffuse reflectance spectroscopy (DRS) and imaging are increasingly being used in surgical guidance for tumor margin detection during endoscopic operations. However, the accuracy of the boundary detection with optical techniques may depend on the acquisition parameters, and its evaluation is in high demand. In this work, using optical phantoms with homogeneous and heterogeneous distribution of chromophores mimicking normal and pathological bladder tissues, the accuracy of tumor margin detection using single-fiber diffuse reflectance spectroscopy and spatial frequency domain imaging was evaluated. We also showed how the diffuse reflectance response obtained at different spatial frequencies with the spatial frequency domain imaging technique could be used not only to quantitatively map absorption and scattering coefficients of normal tissues and tumor-like heterogeneities but also to estimate the tumor depth localization. The demonstrated results could be helpful for proper analysis of the DRS data measured in vivo and for translation of optical techniques for tumor margin detection to clinics.

Mechanical Enhancement and Kinetics Regulation of Fmoc-Diphenylalanine Hydrogels by Thioflavin†T.

The self-assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self-assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc-Diphenylalanine (Fmoc-FF) dipeptide self-assembly using the Thioflavin T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc-FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc-FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro- and macroscale properties.

Ultrafast Energy Transfer Determines the Formation of Fluorescence in DOM and Humic Substances.

Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5-1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor-acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor-acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.

Pericapillary Edema Assessment by Means of the Nailfold Capillaroscopy and Laser Scanning Microscopy.

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.

Assessment of Fibrinogen Macromolecules Interaction with Red Blood Cells Membrane by Means of Laser Aggregometry, Flow Cytometry, and Optical Tweezers Combined with Microfluidics.

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 αIIßß3 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.

The Oxidation-Induced Autofluorescence Hypothesis: Red Edge Excitation and Implications for Metabolic Imaging.

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.

Amplification of photoacoustic effect in bimodal polymer particles by self-quenching of indocyanine green.

A new type of bimodal contrast agent was made that is based on the self-quenching of indocyanine green (ICG) encapsulated in a biocompatible and biodegradable polymer shell. The increasing of a local ICG concentration that is necessary for the obtaining of self-quenching effect was achieved by freezing-induced loading and layer-by-layer assembly. As a result, an efficient photoacoustic(optoacoustic)/fluorescent contrast agent based on composite indocyanine green/polymer particles was successfully prepared and was characterized by fluorescence and photoacoustic(optoacoustic) tomography in vitro. This type of contrast agent holds good promise for clinical application owing to its high efficiency and biosafety.

Label-free characterization of white blood cells using fluorescence lifetime imaging and flow-cytometry: molecular heterogeneity and erythrophagocytosis [Invited].

Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types - white blood cells, red blood cells, erythrophagocytic cells - are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.

In vivo optical imaging of the viable epidermis around the nailfold capillaries for the assessment of heart failure severity in humans.

Heart failure (HF) is among the socially significant diseases, involving over 2% of the adult population in the developed countries. Diagnostics of the HF severity remains complicated due to the absence of specific symptoms and objective criteria. Here, we present an indicator of the HF severity based on the imaging of tissue parameters around the nailfold capillaries. High resolution nailfold video capillaroscopy was performed to determine the perivascular zone (PZ) size around nailfold capillaries, and 2-photon tomography with fluorescence lifetime imaging was used to investigate PZ composition. We found that the size of PZ around the nailfold capillaries strongly correlates with HF severity. Further investigations using 2-photon tomography demonstrated that PZ corresponds to the border of viable epidermis and it was suggested that the PZ size variations were due to the different amounts of interstitial fluid that potentially further translates in clinically significant oedema. The obtained results allow for the development of a quantitative indicator of oedematous syndrome, which can be used in various applications to monitor the dynamics of interstitial fluid retention. We therefore suggest PZ size measured with nailfold video capillaroscopy as a novel quantitative sensitive non-invasive marker of HF severity.