Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

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.

Forces of interaction of red blood cells and endothelial cells at different concentrations of fibrinogen: Measurements with laser tweezers in vitro.

Blood microrheology depends on the constituents of blood plasma, the interaction between blood cells resulting in red blood cell (RBC) and platelets aggregation, and adhesion of RBC, platelets and leukocytes to vascular endothelium. The main plasma protein molecule -actuator of RBC aggregation is fibrinogen. In this paper the effect of interaction between the endothelium and RBC at different fibrinogen concentrations on the RBC microrheological properties was investigated in vitro. Laser tweezers were used to measure the RBC-endothelium interaction forces. It was shown for the first time that the interaction forces between RBC and endothelium are comparable with the RBC aggregation forces, they increase with fibrinogen concentration and reach the saturation level of about 4 pN at the concentration of 4 mg/ml. These results are important for better understanding the mechanisms of RBC and endothelium interaction and developing the novel therapeutic protocols of the microrheology correction in different pathologies.

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.

Effect of size of TiO(2) nanoparticles applied onto glass slide and porcine skin on generation of free radicals under ultraviolet irradiation.

Titanium dioxide (TiO(2)) nanoparticles are extensively used today in sunscreens and coatings as protective compounds for human skin and material surfaces from UV radiation. In this paper, such particles are investigated by electron paramagnetic resonance spectroscopy as sources of free radicals under UV irradiation. The surface density of a placebo with embedded particles corresponds to the recommendations of dermatologists (2 mg cm(-2)). It is revealed that if applied onto glass, small particles 25 nm in diameter produce an increased amount of free radicals compared to the larger ones of 400 nm diam and the placebo itself. However, if applied onto porcine skin in vitro, there is no statistically distinct difference in the amount of radicals generated by the two kinds of particles on skin and by the skin itself. This proves that although particles as part of sunscreens produce free radicals, the effect is negligible in comparison to the production of radicals by skin in vitro.

The Effects of Different Signaling Pathways in Adenylyl Cyclase Stimulation on Red Blood Cells Deformability.

Signaling pathways of red blood cells' (RBCs) micromechanics regulation, which are responsible for maintaining microcirculation, constitute an important property of RBC physiology. Selective control over these processes may serve as an indispensable tool for correction of hemorheological disorders, which accompany a number of systemic diseases (diabetes mellitus I&II, arterial hypertension, malaria, etc.). Activation of certain pathways involving adenylyl cyclase may provide fast adaptive regulation of RBC deformability (RBC-D). However the specific molecular conditions of intracellular signal transduction in mediating RBC microrheological properties at adenylyl cyclase stimulation remain unclear. In this paper, we present the results of the in vitro study of the effects of different signaling pathways in adenylyl cyclase stimulation on RBC-D. We studied (1) the direct stimulation of adenylyl cyclase with forskolin; (2) non-selective adrenoreceptor stimulation with epinephrine; (3) ß2-adrenoreceptor agonist metaproterenol; (4) membrane-permeable analog of cAMP (dibutyryl-cAMP). Using laser ektacytometry, we observed a concentration-dependent increase in RBC-D for all studied effectors. The EC50 values for each substance were estimated to be in the range of 1-100 µM depending on the shear stress applied to the RBC suspension. The results can serve as an evidence of adenylyl cyclase signaling cascade involvement in the regulation of RBC micromechanical properties presenting a complex molecular pathway for fast increase of microcirculation efficiency in case of corresponding physiologic metabolic demands of the organism, e.g., during stress or physical activity. Further studies of this molecular system will reveal new knowledge which may improve the quality of medical treatment of hemorheological disorders.

Optical assessment of alterations of microrheologic and microcirculation parameters in cardiovascular diseases.

In this work, we compare the blood aggregation parameters measured in vitro by laser aggregometry and optical trapping techniques in blood samples with the parameters of blood rheology measured in vivo by digital capillaroscopy in the nail bed capillaries of patients suffering from the hypertension and coronary heart disease. We show that the alterations of the parameters measured in vivo and in vitro for patients with different stages of these diseases are interrelated. Good agreement between the results obtained with different techniques, and their applicability for the diagnostics of abnormalities of rheological properties of blood are demonstrated.

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.

Assessment of the "cross-bridge"-induced interaction of red blood cells by optical trapping combined with microfluidics.

Red blood cell (RBC) aggregation is an intrinsic property of the blood that has a direct effect on the blood viscosity and circulation. Nevertheless, the mechanism behind the RBC aggregation has not been confirmed and is still under investigation with two major hypotheses, known as "depletion layer" and "cross-bridging." We aim to ultimately understand the mechanism of the RBC aggregation and clarify both models. To measure the cell interaction in vitro in different suspensions (including plasma, isotonic solution of fibrinogen, isotonic solution of fibrinogen with albumin, and phosphate buffer saline) while moving the aggregate from one solution to another, an approach combining optical trapping and microfluidics has been applied. The study reveals evidence that RBC aggregation in plasma is at least partly due to the cross-bridging mechanism. The cell interaction strength measured in the final solution was found to be significantly changed depending on the initial solution where the aggregate was formed.

Effect of shear-induced platelet activation on red blood cell aggregation.

Mechanical shear stress is one of the important factors for platelet activation. Although shear stress has been frequently utilized in many applications of diagnostic bio-equipment, there has been little consideration as to whether shear stress induces platelet activation and consequently alters hemorheological characteristics. Therefore, we investigated the effect of shear-induced platelet activation on red blood cell (RBC) aggregation. The hypothesis of the present research is as follows: Platelets activated by high shear stress secrete substances, which can affect hemorheological characteristics to promote RBC aggregation. In our study, an increase in RBC aggregation indices (critical shear stress (CSS) and aggregation index (AI)) by shear-induced platelet activation was observed. Significantly, an increase of 19% in CSS was observed. However, deformability remained unchanged. These phenomena could be a result of the increased cellular adhesion force on RBC membranes due to secreted substances from activated platelets. Therefore, since high shear application results in the unexpected effect on RBC aggregation, conditions for shear application in diagnostic bio-equipment are to be carefully determined.

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization.

The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions.

Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

Effect of size of TiO2 nanoparticles embedded into stratum corneum on ultraviolet-A and ultraviolet-B sun-blocking properties of the skin.

Recently there has been a strong demand to protect human skin against negative effects of the UV solar light. This problem is interesting due to the increased frequency of human diseases caused by such radiation. We aim to evaluate how the optical properties of the horny layer of skin can be effectively changed by imbedding TiO2 fine particles to achieve the maximal attenuation of the UV solar radiation. In-depth distribution of TiO2 particles embedded into the skin by multiple administration of sunscreens is determined experimentally using the tape-stripping technique. A computer code implementing the Monte Carlo method is developed to simulate photon migration within the 20-microm-thick horny layer filled with nanosized TiO2 spheres, 25 to 200 nm in diameter. Dependencies of the UV radiation of two wavelengths (310 and 400 nm) absorbed by and totally reflected from, as well as transmitted through the horny layer on the size of TiO2 particles are obtained and analyzed. The most attenuating particles are found to be 62 and 122 nm in diameter for 310- and 400-nm light, respectively. The former could be suggested as the main fraction to be used in sunscreens to prevent erythema.

Raman microspectroscopy of nanodiamond-induced structural changes in albumin.

Nanodiamonds (NDs) are promising agents for theranostic applications due to reported low toxicity and high biocompatibility, which is still being extensively tested on cellular, tissue, and organism levels. It is presumed that for experimental and future clinical applications, NDs will be administered into the organism via the blood circulation system. In this regard, the interaction of NDs with blood components needs to be thoroughly studied. We studied the interaction of carboxylated NDs (cNDs) with albumin, one of the major proteins of blood plasma. After 2-h long in vitro incubation in an aqueous solution of the protein, 100-nm cNDs were dried and the dry samples were studied with the aid of Raman microspectroscopy. The spectroscopic data indicate significant conformational changes that can be due to cND­protein interaction. A possible decrease in the functional activity of albumin related to the conformational changes must be taken into account in the in vivo applications.

Designing inorganic light-protective skin nanotechnology products.

In this review, we discuss the use of inorganic nanoparticles, mainly zinc oxide (ZnO) and titanium dioxide (TiO2), for sunscreen applications considering their intrinsic physical properties and the Mie theory. These properties cause, from one side, attenuation of the ultraviolet light by absorption and scattering (dependent on a particle size), which is the purpose sunscreens are designed for, and formation of free radicals (i.e., phototoxicity) during this process--from the other. Particle penetration into skin is also an important issue addressed in this review due to possible adverse effects associated with interaction between nanoparticles and skin living cells.

Doppler OCT imaging of cytoplasm shuttle flow in Physarum polycephalum.

The Doppler optical coherence tomography technique was applied to image the oscillatory dynamics of protoplasm in the strands of the plasmodium of slime mould Physarum polycephalum. Radial contractions of the gel-like walls of the strands and the velocity distributions in the sol-like endoplasm streaming along the plasmodial strands are imaged. The motility inhibitor effect of carbon dioxide on the cytoplasm shuttle flow and strand-wall contraction is shown. The optical attenuation coefficient of cytoplasm is estimated.