rus
Issue #3-4/2023
A.A.Truhova, A.I.Akhmetova, I.V.Yaminsky
3D VISUALIZATION OF ERYTHROCYTES BY ATOMIC FORCE MICROSCOPY
3D VISUALIZATION OF ERYTHROCYTES BY ATOMIC FORCE MICROSCOPY
DOI: https://doi.org/10.22184/1993-8578.2023.16.3-4.180.184
Erythrocytes are one of the favorite objects of study in probe microscopy. First, they are easily accessible, do not require long and complex sample preparation, and, most importantly, they are replete with distinctive features that can be used in clinical diagnostics. When red blood cells circulate in the blood, they need to pass through narrow capillary openings much smaller than their own cross-sectional size. The elastic properties of the membrane allow it to pass through the bloodstream and deliver the necessary substances. The ratio of surface area to volume, the viscosity of the cytoplasm, and the internal deformability of membranes affect the ability of red blood cells to transform and pass through narrow spaces. Therefore, the morphological, structural and physical characteristics of blood cells are becoming increasingly important in the study of various diseases or in assessing the risk of their development.
Erythrocytes are one of the favorite objects of study in probe microscopy. First, they are easily accessible, do not require long and complex sample preparation, and, most importantly, they are replete with distinctive features that can be used in clinical diagnostics. When red blood cells circulate in the blood, they need to pass through narrow capillary openings much smaller than their own cross-sectional size. The elastic properties of the membrane allow it to pass through the bloodstream and deliver the necessary substances. The ratio of surface area to volume, the viscosity of the cytoplasm, and the internal deformability of membranes affect the ability of red blood cells to transform and pass through narrow spaces. Therefore, the morphological, structural and physical characteristics of blood cells are becoming increasingly important in the study of various diseases or in assessing the risk of their development.
Теги: bionanoscopy erythrocytes scanning probe microscopy бионаноскопия сканирующая зондовая микроскопия эритроциты эхиноциты
INTRODUCTION
Atomic force microscopy has established itself as an excellent tool for investigating the structural and morphological features of red blood cells. For erythrocytes, atomic force microscopy provides a number of important cell characteristics, and a number of their characteristics were automatically calculated in special software. Using AFM, morphometric portraits of blood cells can be obtained [1].
AFM makes it possible to study the morphological, nanostructural, cytoskeletal and mechanical properties of erythrocytes exposed to various physical and chemical factors, namely hemin, zinc ions and long-term storage [2]. Based on the experimental data obtained, a set of important biomarkers determining condition of blood cells was identified. The damaging effect of cadmium salts on erythrocyte membrane was studied by atomic force microscopy methods [3].
In [4], erythrocytes of patients with megaloblastic anaemia (MA) with hemolysis (destruction of erythrocytes with the release of hemoglobin into the blood plasma) were examined: the average cell surface roughness and surface area in the experimental group were significantly lower than in controls, and the MCV value in patients with MA was higher than in healthy subjects, which means a significant decrease in the surface area to volume ratio and reduced deformability. Such erythrocytes cannot withstand the shear stresses of the arterial circulation. Researchers have hypothesised that reduced cellular deformability prevents preservation of cell integrity during microcirculation and ultimately leads to hemolysis.
In [5], erythrocyte morphology and biochemical parameters were assessed in four groups: healthy individuals, people with prediabetes (PDG), those with metabolic syndrome (MSG) and the diabetic group (DMG). The following morphological parameters were assessed: height, axis ratio, disc concavity depth, thickness, which were also related to age, glycated hemoglobin (HbA1c) values, triglycerides, body mass index, waist-to-hip ratio and physical inactivity.
AFM has shown that sickle-shaped red blood cells have a rougher surface and greater stiffness than normal red blood cells [6].
Considering the above-mentioned studies, the following parameters can be distinguished:
MCV – mean cell volume, the average volume of one erythrocyte;
membrane surface roughness – distinguish between average Ra and Rq (RMS);
deformability – ratio of surface area to volume.
The coefficient of cell flattening (Q) is the ratio of projection area to height. This coefficient characterizes the cells plasticity degree [7].
All the above mentioned parameters are well investigated by AFM.
MATERIALS AND METHODS
As part of this work, human erythrocytes were examined by AFM in contact mode in air on a glass surface using CSG10 cantilever. Atomic force microscopy was performed on a FemtoScan probe microscope. Frame acquisition time of about 8 min.
The results were processed in FemtoScan Online software, which allows to obtain three-dimensional images, construction of contour lengths, filtering, image processing and making the necessary quantitative calculations: area, volume, perimeter, greatest height, roughness of the object [8, 9].
RESULTS
3D images of erythrocytes were obtained for several weeks after having had a coronavirus infection (Fig.1, 2), and were then compared with a sample from a healthy person (Fig.3).
All cells are oval in shape, there are no biconcave disc-shaped cells, all cells are rounded, and surface morphology has a characteristic relief with prominences averaging about 100–150 nm in height.
The overall cell size differs from the normal red cell portrait in AFM – the total cell area and perimeter are reduced, but the volume is increased (the average values for the 6 cells for each case are shown in Table 1).
See also the image of red blood cells from a healthy donor on Fig.3.
DISCUSSION AND CONCLUSIONS
Almost all cells in the sample tested are in the form of echinocytes, although in cell samples from a healthy donor the number of echinocytes in a similar field of view is minimal or not observed at all. Most likely, transformation into echinocytes occurs during sample preparation, suggesting that erythrocytes are sensitive to environmental changes and therefore changes occur rapidly during swabbing and specimen drying on the glass. In healthy donors this process is much slower and the cells retain their biconcave disk shape.
In [10] was examined COVID19 patients blood, it was shown that in the blood of 80.6% of patients both at admission and at discharge there was a marked transformation of part of erythrocytes into echinocytes; proportion of echinocytes in patients at admission was 17.9±3.6% of the total number of observed red cells, at discharge it decreased to 12.1±2.1%.
Atomic force microscopy can be used to see the morphological and structural features of blood cells. It is particularly useful when these parameters can be linked to specific factors or a diagnosis.
ACKNOWLEDGMENTS
The authors are grateful to the Foundation for the Promotion of Innovation (project no. 71108). The study of A.I.Akhmetova was supported by the Russian Science Foundation (project No. 23-74-30003).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Atomic force microscopy has established itself as an excellent tool for investigating the structural and morphological features of red blood cells. For erythrocytes, atomic force microscopy provides a number of important cell characteristics, and a number of their characteristics were automatically calculated in special software. Using AFM, morphometric portraits of blood cells can be obtained [1].
AFM makes it possible to study the morphological, nanostructural, cytoskeletal and mechanical properties of erythrocytes exposed to various physical and chemical factors, namely hemin, zinc ions and long-term storage [2]. Based on the experimental data obtained, a set of important biomarkers determining condition of blood cells was identified. The damaging effect of cadmium salts on erythrocyte membrane was studied by atomic force microscopy methods [3].
In [4], erythrocytes of patients with megaloblastic anaemia (MA) with hemolysis (destruction of erythrocytes with the release of hemoglobin into the blood plasma) were examined: the average cell surface roughness and surface area in the experimental group were significantly lower than in controls, and the MCV value in patients with MA was higher than in healthy subjects, which means a significant decrease in the surface area to volume ratio and reduced deformability. Such erythrocytes cannot withstand the shear stresses of the arterial circulation. Researchers have hypothesised that reduced cellular deformability prevents preservation of cell integrity during microcirculation and ultimately leads to hemolysis.
In [5], erythrocyte morphology and biochemical parameters were assessed in four groups: healthy individuals, people with prediabetes (PDG), those with metabolic syndrome (MSG) and the diabetic group (DMG). The following morphological parameters were assessed: height, axis ratio, disc concavity depth, thickness, which were also related to age, glycated hemoglobin (HbA1c) values, triglycerides, body mass index, waist-to-hip ratio and physical inactivity.
AFM has shown that sickle-shaped red blood cells have a rougher surface and greater stiffness than normal red blood cells [6].
Considering the above-mentioned studies, the following parameters can be distinguished:
MCV – mean cell volume, the average volume of one erythrocyte;
membrane surface roughness – distinguish between average Ra and Rq (RMS);
deformability – ratio of surface area to volume.
The coefficient of cell flattening (Q) is the ratio of projection area to height. This coefficient characterizes the cells plasticity degree [7].
All the above mentioned parameters are well investigated by AFM.
MATERIALS AND METHODS
As part of this work, human erythrocytes were examined by AFM in contact mode in air on a glass surface using CSG10 cantilever. Atomic force microscopy was performed on a FemtoScan probe microscope. Frame acquisition time of about 8 min.
The results were processed in FemtoScan Online software, which allows to obtain three-dimensional images, construction of contour lengths, filtering, image processing and making the necessary quantitative calculations: area, volume, perimeter, greatest height, roughness of the object [8, 9].
RESULTS
3D images of erythrocytes were obtained for several weeks after having had a coronavirus infection (Fig.1, 2), and were then compared with a sample from a healthy person (Fig.3).
All cells are oval in shape, there are no biconcave disc-shaped cells, all cells are rounded, and surface morphology has a characteristic relief with prominences averaging about 100–150 nm in height.
The overall cell size differs from the normal red cell portrait in AFM – the total cell area and perimeter are reduced, but the volume is increased (the average values for the 6 cells for each case are shown in Table 1).
See also the image of red blood cells from a healthy donor on Fig.3.
DISCUSSION AND CONCLUSIONS
Almost all cells in the sample tested are in the form of echinocytes, although in cell samples from a healthy donor the number of echinocytes in a similar field of view is minimal or not observed at all. Most likely, transformation into echinocytes occurs during sample preparation, suggesting that erythrocytes are sensitive to environmental changes and therefore changes occur rapidly during swabbing and specimen drying on the glass. In healthy donors this process is much slower and the cells retain their biconcave disk shape.
In [10] was examined COVID19 patients blood, it was shown that in the blood of 80.6% of patients both at admission and at discharge there was a marked transformation of part of erythrocytes into echinocytes; proportion of echinocytes in patients at admission was 17.9±3.6% of the total number of observed red cells, at discharge it decreased to 12.1±2.1%.
Atomic force microscopy can be used to see the morphological and structural features of blood cells. It is particularly useful when these parameters can be linked to specific factors or a diagnosis.
ACKNOWLEDGMENTS
The authors are grateful to the Foundation for the Promotion of Innovation (project no. 71108). The study of A.I.Akhmetova was supported by the Russian Science Foundation (project No. 23-74-30003).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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