Currently, much research is devoted to the study of biological objects using atomic force microscopy (AFM). This method’s resolution is superior to the other non-scanning techniques. Our study aims to further emphasize some of the advantages of using AFM as a clinical screening tool. The study focused on red blood cells exposed to various physical and chemical factors, namely hemin, zinc ions, and long-term storage. AFM was used to investigate the morphological, nanostructural, cytoskeletal, and mechanical properties of red blood cells (RBCs). Based on experimental data, a set of important biomarkers determining the status of blood cells have been identified.

Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.

Iron is needed for life-essential processes, but free iron overload causes dangerous clinical consequences. The study of the role of red blood cells (RBCs) in the influence of excess free iron in the blood on the pathological consequences in an organism is relevant. Here, in a direct biophysical experiment in vitro, we studied the action of free iron overload on the packed red blood cell (pRBC) characteristics. In experiments, we incubated pRBCs with the ferrous sulfate solution (Fe²⁺). Wе used free iron in a wide range of concentrations. High concentrations made us possible to establish the pattern of the toxic effect of excess iron on pRBCs during a reduced incubation time in a biophysical experiment in vitro. It was found that excess free iron causes changes in pRBC morphology, the appearance of bridges between cells, and the formation of clots, increasing the membrane stiffness and methemoglobin concentration. We created a kinetic model of changes in the hemoglobin derivatives. The complex of simultaneous distortions of pRBCs established in our experiments can be taken into account when studying the mechanism of the toxic influence of excess free iron in the blood on pathological changes in an organism.

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.

The impact of carbon monoxide (CO) gas on the human organism is very dangerous. The affinity of CO to hemoglobin is considerably higher than that of oxygen. Thus, the interaction of CO with the blood results in a higher content of carboxyhemoglobin (HbCO) in red blood cells (RBCs) and correspondingly in tissue hypoxia. The disruption in the organism depends on the HbCO content in the blood. To assess any complications in the body at a given moment due to CO exposure and predict future consequences, it is necessary to measure the dynamics of hemoglobin derivative concentrations simultaneously. However, measuring HbCO and other derivatives in RBCs without hemolysis accurately is complicated due to the strong intercollinearity between the molar absorptivities of hemoglobin derivatives and superposition of absorption and scattering spectra. In the present study, to quantitatively assess the contents of the hemoglobin derivatives in the blood after exposure to CO, improved accuracy is achieved by optimizing the wavelength range used for the nonlinear curve fitting of optical spectra. Experimental spectra were measured in the wavelength range Δλ=500−700nm. For each experimental curve, it was established the value of optimal interval Δλopt for which the correlation coefficient between experimental data and corresponding points of the theoretical fitting curve was the maximum in the wavelength range Δλtyp=535−580nm, which contains the typical absorption peaks for HbO2, Hb, and HbCO. The concentrations obtained based on such fitting curves were considered to be highly accurate. The quantitative assessment enabled the determination of theHbCO nonlinear increase with the time of CO exposure in the in vitro experiment and the study of the dynamics of hemoglobin derivative transformations during blood incubation.

The influences of various factors on blood lead to the formation of extra reactive oxygen species (ROS), resulting in the disruption of morphology and functions of red blood cells (RBCs). This study considers the mechanisms of the mechanochemical synergism of $O{H}^{•}$ free radicals, which are most active in the initiation of lipid peroxidation (LPO) in RBC membranes, and $H_2{O}_2$ molecules, the largest typical diffusion path. Using kinetic models of differential equations describing $C_{H_2{O}_2}\left(t\right)$ and $C_{O{H}^{•}}\left(t\right)$, we discuss two levels of mechanochemical synergism that occur simultaneously: (1) synergism that ensures the delivery of highly active free radicals $O{H}^{•}$ to RBC membranes and (2) a positive feedback system between $H_2{O}_2$ and $O{H}^{•}$, resulting in the partial restoration of spent molecules. As a result of these ROS synergisms, the efficiency of LPO in RBC membranes sharply increases. In blood, the appearance of $O{H}^{•}$ free radicals is due to the interaction of $H_2{O}_2$ molecules with free iron ions ($F{e}^{2+}$) which arise as a result of heme degradation. We experimentally established the quantitative dependences of $C_{O{H}^{•} }\left(C_{H_2{O}_2}\right)$ using the methods of spectrophotometry and nonlinear curve fitting. This study extends the analysis of the influence of ROS mechanisms in RBC suspensions.