rus
Issue #5/2025
A.I.Akhmetova, I.V.Yaminsky
MORPHOLOGY OF BLOOD CELLS BASED ON ATOMIC FORCE MICROSCOPY DATA
MORPHOLOGY OF BLOOD CELLS BASED ON ATOMIC FORCE MICROSCOPY DATA
Probe microscopy allows us to study the morphology, nanostructure of the membrane, mechanical properties and biochemical interactions of blood cells over time in liquid and in air. The mechanical properties, rigidity and elasticity of the membrane can be quantified using atomic force microscopy (AFM). AFM can be used in a variety of areas: from assessing the quality of stored blood in transfusion banks to elucidating the molecular mechanisms of oxidative damage and disease-related changes. The use of AFM in the study of red blood cells helps in understanding the causes of neurodegenerative diseases, diabetes, and miscarriages.
INTRODUCTION
The erythrocyte is a cell consisting of cytoplasm and a cell membrane, which can be divided into two parts: a lipid bilayer and a cytoskeleton. The cytoskeleton interacts with integral proteins and lipids to maintain membrane integrity. It plays an important role in maintaining shape, ensuring cell flexibility, and organizing lipids in the erythrocyte. A deficiency of any of the proteins can lead to changes in the structure of the erythrocyte cytoskeleton, which reduces the overall degree of membrane elasticity. This, in turn, may lead to the inability of the erythrocyte to change shape when passing through capillaries, causing microvascular complications and dysfunctions in oxygen transport.
Changes in erythrocyte morphology are associated with the aging of erythrocytes and pathological conditions. AFM allows obtaining high-resolution images of erythrocyte surfaces without the need for complex sample preparation, without fixation, in conditions close to natural, such as in buffered solutions (Fig.1). This allows visualization of the smallest surface structures, including the arrangement of nanometer-scale particles on the erythrocyte membrane, with lateral resolution of 0.5–1 nm and vertical resolution of 0.1–0.2 nm (Fig. 2) [1].
Studies have shown that during prolonged storage (up to 35 days at +4 °C), the diameter of blood cells decreases, while the Young’s modulus of erythrocyte membranes significantly increases, which correlates with morphological changes: transformation of discocytes into echinocytes (Fig.3) and spheroechinocytes, which are less suitable for blood transfusion purposes [2]. The average values and the width of distribution of the Young’s modulus significantly increased in samples taken from patients with diabetes and smokers compared to samples taken from healthy donors; at the same time, the average values of the Young’s modulus increase with the age of the patient.
In the work [4], normal aging of erythrocytes was visualized as transformations in cell shape (from normal biconcave discs through various morphological classes to complete loss of normal shape, i.e., dense spherocytes). All these changes were accompanied by a gradual decrease in the size and roughness of the cell membranes. Reduction in diameter was associated with a change in shape. Using AFM, it was established that the diameter of biconcave and serrated cells was larger than that of spiculated and spherical erythrocytes. Increasing of populations of spiculocytes and spherocytes occurred significantly earlier in women suffering from early-stage miscarriages (EMS) compared to the control groups (healthy women and pregnant women). Different pathways of red blood cell aging were demonstrated, with a faster onset of morphological changes in red blood cells during EMS compared to both control groups, and a difference in membrane roughness of fresh red blood cells during EMS compared to red blood cells from the two control groups. The significantly higher value of the root mean square roughness of the cells in the EMS group is due to a more uneven folding of the membrane and appearance of certain protrusions, likely due to disruption in plasma membrane integrity and/or the onset of microvesiculation. Vesiculation of mature red blood cells helps to remove defective areas of the erythrocyte membrane. It is known that in certain pathological situations and during the aging of erythrocytes, vesiculation of the plasma membrane is enhanced [5, 6]. The release of microvesicles (micro-particles) occurs as a result of redistribution of phosphatidylserine and phosphatidylethanolamine from the inner leaflet to the outer leaflet of the plasma membrane and occurs selectively in lipid-rich microdomains within the plasma membrane [7].
The change in cell shape due to partial or complete loss of deformability can occur as a result of various pathological conditions [8], and accumulation of reactive oxygen species (ROS), or as part of the aging process of erythrocytes [9]. Decreased elasticity is a sign of reduced cell lifespan and can be caused by various factors related to oxidative stress, altered membrane composition, and pathological conditions.
It is known that the shape of red blood cells is largely determined by the mechanical properties of the cytoskeleton and abundance of adenosine triphosphate (ATP) [10]. ATP is important for maintaining the disc shape of red blood cells, as depletion of ATP causes a change from discocyte to echinocyte form [11]. For example, sickle-shaped erythrocytes are characterized by elevated oxidative stress levels and lower ATP levels [22]. It has been shown that in pregnant women, the ATP level in erythrocytes is significantly lower than in non-pregnant women [13].
Increased membrane stiffness is characteristic of erythrocytes obtained from patients with neurodegenerative diseases compared to healthy individuals. In the study [14], using atomic force and traditional optical microscopy, ultrastructural analysis of erythrocytes was conducted as a model of peripheral cells with characteristic signs for neurodegenerative pathologies - Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). Blood cells in neurodegenerative pathologies are characterized by a reduced number of biconcave disc-shaped cells, lower surface roughness, and a higher Young’s modulus compared to healthy cells. The biconcave shape remains the predominant form in cells in ALS and AD, while jagged cells dominate the morphology in PD.
It has been experimentally established that gemin and oxidative processes in the blood have a specific effect on the erythrocyte membranes nanostructure, forming domains on the surface. The characteristic size of the granular structures in the domains is 100-200 nm, which coincides with the characteristic size of the spectrin matrix [15].
Study of oxidative and redox processes
AFM is effectively used to study changes at the molecular level in erythrocytes caused by oxidation. Oxidative stress induced by ultraviolet (UV) radiation disrupts the spectrin network of the cytoskeleton underlying the erythrocyte membrane, leading to morphological changes and loss of the typical discocyte shape. The size of the spectrin network cells increases from approximately 80–200 nm in control samples to 600–1000 nm after UV radiation exposure, indicating structural breakdown [4, 5, 16, 17]. Treatment with antioxidants may preserve integrity of the spectrin network and morphology of erythrocytes, as confirmed by AFM studies.
Using AFM, changes in leukocytes morphology in leukemia were identified – spike-like protrusions appeared on the surface [18]. Upon closer examination of the images of leukocytes, arrangement on the substrate resembles sludge. White blood cells from diabetic patients were stiffer than leukocytes from healthy donors. Compared to healthy donors, the diameter of the leukocytes and the Young’s modulus were significantly increased, although the height of the cells decreased [19].
Erythrocytes and leukocytes in patients with diabetes show changes in shape, increased stiffness, and a tendency to aggregate. This study on the effects of diabetes on erythrocytes demonstrates that excess glucose in the blood leads to significant deformation of erythrocyte shape and slight corrosion on their surface, as glucose can pass through the membrane and encase it like a shell. Red blood cells increase in size and stick together, ultimately leading to increased viscosity and slowed mobility of red blood cells [20]. This study also compared the effects of diabetes and smoking on parameters such as diameter, pit depth, and roughness of red blood cells. It turned out that smoking has a significantly stronger effect on the changes of all three parameters than diabetes: for heavy smokers, roughness increases by 5 times, pit depth decreases by 3.3 times, and red blood cells take on an irregular shape.
The AFM study of blood cells in diabetes revealed a difference in morphology: erythrocytes from the healthy group are normocytes: round, with a visible indentation; erythrocytes from the prediabetes group have an irregular shape, elongated erythrocytes, and indentation is not in the center. Images of erythrocytes from patients with metabolic syndrome resemble stomatocytes. The group of patients with type 2 diabetes has an abnormal morphology of erythrocytes (“crested” erythrocytes) [21].
A study of blood cells in thalassemia showed that erythrocytes become jagged, have various morphologies, are anisocytotic, and take on a hypochromic form. Only a few cells are represented in the form of a biconcave disc. The cells are smaller in size and deformed, with formation of numerous large holes. Erythrocytes in iron deficiency anemia have an even more deformed morphology than in thalassemia: the cells are presented in the form of elliptoids, round, oval, and there is greater swelling in the center of the erythrocytes [22].
Using AFM, it was discovered that the red blood cells of patients with megaloblastic anemia had a larger size; however, their height, average roughness, peak-to-valley value Rp-v (which represents the difference between the maximum and minimum height along the Z-axis on the cell surface in the area of analysis), and surface area were lower compared to the control group [23].
In patients in the department of anesthesiology and intensive care with traumatic brain injury, ischemic and hemorrhagic stroke, cerebral edema, and post-hypoxic encephalopathy, various forms of blood cells were observed, and local defects appeared on the surface of the membranes of erythrocytes: defects of pallor, torus, and nanostructures. A number of defects serve as the starting mechanisms for development of total membrane damage. In all patients included in the study, 16±7% of active platelets were observed on the smear. Platelets are among the indicators of the acute phase of inflammation in sepsis, tumors, and hemorrhages. An increase in the number of platelets is a signal for development of inflammatory pathologies [24].
Infectious diseases (malaria, COVID-19, HIV)
The work [25] discovered a change in the erythrocytes geometry: an increase of 14% in diameter and a decrease of 30% in height compared to the control group. Using AFM, changes in the erythrocytes structure were identified in COVID-19: an increase in cell diameter from 9 to 10.5 micrometers, a noticeable decrease in the height of the dip in the center of the disk, and an increase in the Young’s modulus from 4.5 to 8 kPa.
Using AFM, abnormal forms of erythrocytes were recorded, such as acanthocytes [26]. Echinocytes were also found in the blood smear after COVID-19 [27]. In malaria, parasites alter the erythrocyte membrane, forming protrusions that promote adhesion to vessel walls [28]. Sharp changes in the spectrin network were also found on the surface after infection, i.e., the spectrin network is partially destroyed at a certain stage of infection.
CONCLUSIONS
This review summarizes the results of recent studies demonstrating the capabilities of AFM in study of erythrocytes and their application in clinical practice. Elasticity of the erythrocyte membrane is a very important factor in determining health status. Quantitative determination is crucial for understanding the mechanisms leading to the disruption of red blood cell elasticity. AFM methods can also be successfully applied for monitoring therapeutic procedures.
AFM allows for detailed study of the shape, relief, and nanostructure of erythrocyte membranes, revealing changes associated with various physiological and pathological conditions, such as intoxication, diabetes, megaloblastic anemia, and other diseases.
AFM allows for measurement of key morphometric parameters of erythrocytes - size, axial ratio, depth of disc concavity, thickness, and surface roughness. These characteristics reflect deformability and elastic properties of the membrane, which are important for the passage of erythrocytes through narrow capillaries and for maintaining their functional activity in the bloodstream. The Femtoscan Online software toolkit allows for quantitative determination of cell morphological parameters: average and root mean square roughness, assessment of the erythrocyte indentation depth, and also other parameters [29].
In addition, AFM allows for identification of microscopic damage and defects in membranes that may occur due to toxins, stress factors, or during blood storage. This makes AFM a valuable tool for the diagnosis and monitoring of diseases related to changes in morphology and mechanics of erythrocytes.
AFM facilitates morphological and biomechanical changes detection in erythrocytes associated with various blood diseases and systemic conditions. Changes in membrane stiffness and elasticity serve as biomarkers of pathological states, providing advantages over traditional biochemical analyses, which may not be sensitive enough and require larger sample volumes. AFM also allows for visualization of blood cells in situ and in vivo, facilitating early diagnosis and monitoring of disease progression.
Thus, atomic force microscopy provides the comprehensive morphological and structural study of red blood cells, which contributes to a better understanding of their physiology and pathology at the nanometer level.
ACKNOWLEDGEMENTS
This work was performed under the state order of the Lomonosov Moscow State University. FemtoScan Online software is provided by “Advanced Technologies Center”, www.femtoscan.ru
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.
The erythrocyte is a cell consisting of cytoplasm and a cell membrane, which can be divided into two parts: a lipid bilayer and a cytoskeleton. The cytoskeleton interacts with integral proteins and lipids to maintain membrane integrity. It plays an important role in maintaining shape, ensuring cell flexibility, and organizing lipids in the erythrocyte. A deficiency of any of the proteins can lead to changes in the structure of the erythrocyte cytoskeleton, which reduces the overall degree of membrane elasticity. This, in turn, may lead to the inability of the erythrocyte to change shape when passing through capillaries, causing microvascular complications and dysfunctions in oxygen transport.
Changes in erythrocyte morphology are associated with the aging of erythrocytes and pathological conditions. AFM allows obtaining high-resolution images of erythrocyte surfaces without the need for complex sample preparation, without fixation, in conditions close to natural, such as in buffered solutions (Fig.1). This allows visualization of the smallest surface structures, including the arrangement of nanometer-scale particles on the erythrocyte membrane, with lateral resolution of 0.5–1 nm and vertical resolution of 0.1–0.2 nm (Fig. 2) [1].
Studies have shown that during prolonged storage (up to 35 days at +4 °C), the diameter of blood cells decreases, while the Young’s modulus of erythrocyte membranes significantly increases, which correlates with morphological changes: transformation of discocytes into echinocytes (Fig.3) and spheroechinocytes, which are less suitable for blood transfusion purposes [2]. The average values and the width of distribution of the Young’s modulus significantly increased in samples taken from patients with diabetes and smokers compared to samples taken from healthy donors; at the same time, the average values of the Young’s modulus increase with the age of the patient.
In the work [4], normal aging of erythrocytes was visualized as transformations in cell shape (from normal biconcave discs through various morphological classes to complete loss of normal shape, i.e., dense spherocytes). All these changes were accompanied by a gradual decrease in the size and roughness of the cell membranes. Reduction in diameter was associated with a change in shape. Using AFM, it was established that the diameter of biconcave and serrated cells was larger than that of spiculated and spherical erythrocytes. Increasing of populations of spiculocytes and spherocytes occurred significantly earlier in women suffering from early-stage miscarriages (EMS) compared to the control groups (healthy women and pregnant women). Different pathways of red blood cell aging were demonstrated, with a faster onset of morphological changes in red blood cells during EMS compared to both control groups, and a difference in membrane roughness of fresh red blood cells during EMS compared to red blood cells from the two control groups. The significantly higher value of the root mean square roughness of the cells in the EMS group is due to a more uneven folding of the membrane and appearance of certain protrusions, likely due to disruption in plasma membrane integrity and/or the onset of microvesiculation. Vesiculation of mature red blood cells helps to remove defective areas of the erythrocyte membrane. It is known that in certain pathological situations and during the aging of erythrocytes, vesiculation of the plasma membrane is enhanced [5, 6]. The release of microvesicles (micro-particles) occurs as a result of redistribution of phosphatidylserine and phosphatidylethanolamine from the inner leaflet to the outer leaflet of the plasma membrane and occurs selectively in lipid-rich microdomains within the plasma membrane [7].
The change in cell shape due to partial or complete loss of deformability can occur as a result of various pathological conditions [8], and accumulation of reactive oxygen species (ROS), or as part of the aging process of erythrocytes [9]. Decreased elasticity is a sign of reduced cell lifespan and can be caused by various factors related to oxidative stress, altered membrane composition, and pathological conditions.
It is known that the shape of red blood cells is largely determined by the mechanical properties of the cytoskeleton and abundance of adenosine triphosphate (ATP) [10]. ATP is important for maintaining the disc shape of red blood cells, as depletion of ATP causes a change from discocyte to echinocyte form [11]. For example, sickle-shaped erythrocytes are characterized by elevated oxidative stress levels and lower ATP levels [22]. It has been shown that in pregnant women, the ATP level in erythrocytes is significantly lower than in non-pregnant women [13].
Increased membrane stiffness is characteristic of erythrocytes obtained from patients with neurodegenerative diseases compared to healthy individuals. In the study [14], using atomic force and traditional optical microscopy, ultrastructural analysis of erythrocytes was conducted as a model of peripheral cells with characteristic signs for neurodegenerative pathologies - Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). Blood cells in neurodegenerative pathologies are characterized by a reduced number of biconcave disc-shaped cells, lower surface roughness, and a higher Young’s modulus compared to healthy cells. The biconcave shape remains the predominant form in cells in ALS and AD, while jagged cells dominate the morphology in PD.
It has been experimentally established that gemin and oxidative processes in the blood have a specific effect on the erythrocyte membranes nanostructure, forming domains on the surface. The characteristic size of the granular structures in the domains is 100-200 nm, which coincides with the characteristic size of the spectrin matrix [15].
Study of oxidative and redox processes
AFM is effectively used to study changes at the molecular level in erythrocytes caused by oxidation. Oxidative stress induced by ultraviolet (UV) radiation disrupts the spectrin network of the cytoskeleton underlying the erythrocyte membrane, leading to morphological changes and loss of the typical discocyte shape. The size of the spectrin network cells increases from approximately 80–200 nm in control samples to 600–1000 nm after UV radiation exposure, indicating structural breakdown [4, 5, 16, 17]. Treatment with antioxidants may preserve integrity of the spectrin network and morphology of erythrocytes, as confirmed by AFM studies.
Using AFM, changes in leukocytes morphology in leukemia were identified – spike-like protrusions appeared on the surface [18]. Upon closer examination of the images of leukocytes, arrangement on the substrate resembles sludge. White blood cells from diabetic patients were stiffer than leukocytes from healthy donors. Compared to healthy donors, the diameter of the leukocytes and the Young’s modulus were significantly increased, although the height of the cells decreased [19].
Erythrocytes and leukocytes in patients with diabetes show changes in shape, increased stiffness, and a tendency to aggregate. This study on the effects of diabetes on erythrocytes demonstrates that excess glucose in the blood leads to significant deformation of erythrocyte shape and slight corrosion on their surface, as glucose can pass through the membrane and encase it like a shell. Red blood cells increase in size and stick together, ultimately leading to increased viscosity and slowed mobility of red blood cells [20]. This study also compared the effects of diabetes and smoking on parameters such as diameter, pit depth, and roughness of red blood cells. It turned out that smoking has a significantly stronger effect on the changes of all three parameters than diabetes: for heavy smokers, roughness increases by 5 times, pit depth decreases by 3.3 times, and red blood cells take on an irregular shape.
The AFM study of blood cells in diabetes revealed a difference in morphology: erythrocytes from the healthy group are normocytes: round, with a visible indentation; erythrocytes from the prediabetes group have an irregular shape, elongated erythrocytes, and indentation is not in the center. Images of erythrocytes from patients with metabolic syndrome resemble stomatocytes. The group of patients with type 2 diabetes has an abnormal morphology of erythrocytes (“crested” erythrocytes) [21].
A study of blood cells in thalassemia showed that erythrocytes become jagged, have various morphologies, are anisocytotic, and take on a hypochromic form. Only a few cells are represented in the form of a biconcave disc. The cells are smaller in size and deformed, with formation of numerous large holes. Erythrocytes in iron deficiency anemia have an even more deformed morphology than in thalassemia: the cells are presented in the form of elliptoids, round, oval, and there is greater swelling in the center of the erythrocytes [22].
Using AFM, it was discovered that the red blood cells of patients with megaloblastic anemia had a larger size; however, their height, average roughness, peak-to-valley value Rp-v (which represents the difference between the maximum and minimum height along the Z-axis on the cell surface in the area of analysis), and surface area were lower compared to the control group [23].
In patients in the department of anesthesiology and intensive care with traumatic brain injury, ischemic and hemorrhagic stroke, cerebral edema, and post-hypoxic encephalopathy, various forms of blood cells were observed, and local defects appeared on the surface of the membranes of erythrocytes: defects of pallor, torus, and nanostructures. A number of defects serve as the starting mechanisms for development of total membrane damage. In all patients included in the study, 16±7% of active platelets were observed on the smear. Platelets are among the indicators of the acute phase of inflammation in sepsis, tumors, and hemorrhages. An increase in the number of platelets is a signal for development of inflammatory pathologies [24].
Infectious diseases (malaria, COVID-19, HIV)
The work [25] discovered a change in the erythrocytes geometry: an increase of 14% in diameter and a decrease of 30% in height compared to the control group. Using AFM, changes in the erythrocytes structure were identified in COVID-19: an increase in cell diameter from 9 to 10.5 micrometers, a noticeable decrease in the height of the dip in the center of the disk, and an increase in the Young’s modulus from 4.5 to 8 kPa.
Using AFM, abnormal forms of erythrocytes were recorded, such as acanthocytes [26]. Echinocytes were also found in the blood smear after COVID-19 [27]. In malaria, parasites alter the erythrocyte membrane, forming protrusions that promote adhesion to vessel walls [28]. Sharp changes in the spectrin network were also found on the surface after infection, i.e., the spectrin network is partially destroyed at a certain stage of infection.
CONCLUSIONS
This review summarizes the results of recent studies demonstrating the capabilities of AFM in study of erythrocytes and their application in clinical practice. Elasticity of the erythrocyte membrane is a very important factor in determining health status. Quantitative determination is crucial for understanding the mechanisms leading to the disruption of red blood cell elasticity. AFM methods can also be successfully applied for monitoring therapeutic procedures.
AFM allows for detailed study of the shape, relief, and nanostructure of erythrocyte membranes, revealing changes associated with various physiological and pathological conditions, such as intoxication, diabetes, megaloblastic anemia, and other diseases.
AFM allows for measurement of key morphometric parameters of erythrocytes - size, axial ratio, depth of disc concavity, thickness, and surface roughness. These characteristics reflect deformability and elastic properties of the membrane, which are important for the passage of erythrocytes through narrow capillaries and for maintaining their functional activity in the bloodstream. The Femtoscan Online software toolkit allows for quantitative determination of cell morphological parameters: average and root mean square roughness, assessment of the erythrocyte indentation depth, and also other parameters [29].
In addition, AFM allows for identification of microscopic damage and defects in membranes that may occur due to toxins, stress factors, or during blood storage. This makes AFM a valuable tool for the diagnosis and monitoring of diseases related to changes in morphology and mechanics of erythrocytes.
AFM facilitates morphological and biomechanical changes detection in erythrocytes associated with various blood diseases and systemic conditions. Changes in membrane stiffness and elasticity serve as biomarkers of pathological states, providing advantages over traditional biochemical analyses, which may not be sensitive enough and require larger sample volumes. AFM also allows for visualization of blood cells in situ and in vivo, facilitating early diagnosis and monitoring of disease progression.
Thus, atomic force microscopy provides the comprehensive morphological and structural study of red blood cells, which contributes to a better understanding of their physiology and pathology at the nanometer level.
ACKNOWLEDGEMENTS
This work was performed under the state order of the Lomonosov Moscow State University. FemtoScan Online software is provided by “Advanced Technologies Center”, www.femtoscan.ru
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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