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.

Thermal-induced conformational changes and protein–protein interactions of bovine serum albumin (BSA) in aqueous solution are assessed by small angle X-ray scattering (SAXS) at two pH values (7.4 and 9.0) and two ionic strengths (0.1 and 0.5). We demonstrate that Guinier analysis in two ranges of the modulus of the scattering vector allows protein melting and aggregation to be monitored simultaneously, thus providing insights into the mechanism of thermal-induced BSA aggregation. Results of the analysis suggest that at room temperature monomeric and dimeric BSA fractions are present in solution. For low concentrations (<10 mg mL⁻¹) the monomeric to dimeric fraction ratio is close to 6, the same value we obtained independently in size-exclusion chromatography experiments. For elevated concentrations (20 mg mL⁻¹ and 40 mg mL⁻¹) a decrease in the dimer fraction occurs. Following heating, dimer formation is observed prior to protein melting, while no higher order aggregates are observed in the 20–60 °C temperature range. In the vicinity of the BSA melting point, higher order aggregates appear and protein molecules exhibit an aggregation burst. Higher ionic strength makes the described effects more pronounced – dimer formation increases at lower temperatures, presumably due to partial screening of electrostatic interactions between protein molecules. Moreover, the melting temperature shifts to higher values upon increasing the protein concentration and pH, indicating that repulsive interactions stabilize the protein structure. The suggested model was verified by the assessment of parameters of protein–protein interaction potentials based on DLVO theory using the global fitting procedure.

Tyrosine fluorescence in native proteins is known to be effectively quenched, whereas its emission increases upon proteins’ unfolding. This suggests that tyrosine fluorescence could be exploited for probing structural rearrangements of proteins in addition to the extensively used tryptophan emission. We studied the possibility of using tyrosine fluorescence as an indicator of surfactant-induced conformational changes in albumins. It was shown that fluorescence of tyrosine residues, which are uniformly distributed all over the protein molecules, allows the detection of subtle structural rearrangements of proteins upon surfactant binding, which do not influence the properties of a single tryptophan residue buried in the inner hydrophobic region of human serum albumin. Tyrosine fluorescence properties, including its fluorescence lifetime, revealed the multistage character of surfactant binding to albumin, consistent with the data provided by other methods. The obtained results demonstrate the possibility of probing conformational changes in proteins using tyrosine photophysical parameters as indicators.

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.