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DOI: https://doi.org/10.22184/1993-8578.2024.17.3-4.184.189
The study of the morphology of objects and their mechanical characteristics makes it possible to detect the unique properties of cells and associate these features with development under normal conditions or in the presence of pathologies. To measure the surface of a sample, scanning capillary microscopy (SCM) uses an electrolyte-filled capillary with a nano-sized hole at the tip as a probe. The main advantage of SCM is the non-contact visualization of the biological objects topography in the natural environment – scanning is carried out without forceful contact of the probe tip with the sample surface. Additionally, SCM can be used to determine electrical charges at the solid-liquid interface. In this article, we describe the basics of SCM, its capabilities for imaging cells, and measuring the biomechanical properties of living samples.
The study of the morphology of objects and their mechanical characteristics makes it possible to detect the unique properties of cells and associate these features with development under normal conditions or in the presence of pathologies. To measure the surface of a sample, scanning capillary microscopy (SCM) uses an electrolyte-filled capillary with a nano-sized hole at the tip as a probe. The main advantage of SCM is the non-contact visualization of the biological objects topography in the natural environment – scanning is carried out without forceful contact of the probe tip with the sample surface. Additionally, SCM can be used to determine electrical charges at the solid-liquid interface. In this article, we describe the basics of SCM, its capabilities for imaging cells, and measuring the biomechanical properties of living samples.
INTRODUCTION
Scanning capillary microscopy (SCM) is another type of probe microscopy used to obtain three-dimensional topographic information of biological samples in liquid [1]. SCM measures the ionic current through a capillary filled with electrolyte, which strongly depends on the distance between the probe and the sample. The SCM resolution is at the level of nanometre units, slightly losing to atomic force microscopy (AFM), but the non-contact nature of SCM measurements does not damage biological samples, which are characterised by reduced mechanical stiffness. SCM makes it possible not only to obtain static pictures of cells, but also to carry out long-term monitoring of living biological objects without affecting the sample and to get important data on their biomechanical properties. Therefore, scanning capillary microscopy becomes an indispensable tool for study of living systems.
The membrane of living human erythrocytes carries out oscillations of submicron scale, which have been mainly studied by optical microscopy. The functional role of this phenomenon is still unclear; the membrane oscillations amplitude is regarded as an indicator of mechanical resistance to stresses occurring in capillary beds. It is not easy to experimentally measure membrane oscillations; the main challenge is to develop methods that allow tracking very small displacements at a very high rate, preferably over a large area and for a long time [2].
In [3], cell membrane oscillations dynamics in erythrocytes was studied by analysing the ionic current noise recorded near the cell membrane using SCM. A capillary with a radius of about 300 nm collected data on oscillations with an amplitude of about 200 nm at the cell membrane. Ion current noise was recorded at a fixed distance from the cell and used to determine the amplitude of the membrane oscillations. The slope of the current-distance curve was used as a conversion factor. It was found that the amplitude of oscillations decreased with increasing membrane tension caused by different methods. Decrease in osmolarity of the medium caused swelling of cells and decrease in the oscillations amplitude. A similar result was obtained when polylysine concentration was increased a hundredfold: this treatment increased cell adhesion and stiffness. Both cellular and local increases in membrane tension caused a decrease in the cell membrane oscillations amplitude.
Platelet activation plays a crucial role in haemostasis and thrombosis. It is well known that platelets control retraction and compaction of the blood clot, but their mechanical properties have rarely been studied. Scanning capillary microscopy has been used to visualise morphological and mechanical properties of living human platelets with high spatial resolution [4]. Their mean elastic modulus was presented to decrease during thrombin-induced activation by approximately twofold. A similar softening of platelets was observed during cytoskeleton depolymerisation induced by cytochalasin D, and the researchers were able to distinguish between the effects of thrombin and cytochalasin D.
Human mesenchymal stem cells (MSCs) are interesting as a research object for regenerative medicine in various diseases. Osteogenic differentiation is one of the capabilities of MSCs, which is crucial for their clinical applications. Cell deformability and changes in mechanical properties during cell death were recorded using AFM [5, 6]. During adipogenic and osteogenic differentiation [7], topography of MSCs, osteoblasts and osteosarcoma cells were studied using AFM [8]. The results showed that cell height may be an important factor in osteogenic differentiation. With the help of SCM in [9] we quantitatively assessed long-term changes in MSC topography during osteogenic differentiation. It was shown that different cell lines undergo different morphological changes during osteogenic differentiation.
In [10], 3D morphology and roughness of A549 adenocarcinoma cells under physiological conditions before and after cisplatin-induced apoptosis for 24 hours were studied. Tracing morphology of the same single A549 cells exposed to cisplatin revealed heterogeneity of response to the drug, formation of membrane bubbles and increased membrane roughness.
Colorectal cancer is closely related to the level of hydrogen peroxide (H2O2) in the tumour microenvironment. Several clinical studies on the use of H2O2 in cancer treatment have revealed its paradoxical role as a stimulator of cancer progression. A study [11] combined SCM with highly sensitive Pt-functionalised electrodes to measure dynamic extracellular and intracellular H2O2 gradients in individual Caco-2 colorectal cancer cells. The relationship between cell mechanical properties and H2O2 gradients was revealed. Exposure to H2O2 at 0.1 or 1 mmol/L concentration increased the gradient of extracellular and intracellular H2O2. Remarkably, cellular F-actin-dependent stiffness increased at 0.1 mmol/L but decreased when H2O2 eustresses 1 mmol/L. This study shows the complex interplay between physical properties and biochemical signals in the antioxidant defence of tumour cells, demonstrating the use of H2O2 eustress for survival at the level of individual cells. Inhibition of glutathione peroxidase 2 or catalase enhances the cytotoxic activity of H2O2 eustress against colorectal cancer cells, which holds promise for the development of H2O2 related therapies for cancer and other inflammatory diseases.
The layer-by-layer film deposition method is the alternate adsorption of anionic and cationic polyelectrolytes via electrostatic interactions. In order to prepare thin films with desired structure and properties, it is important to understand how different fabrication conditions affect the thin films formation. In [12], they performed in situ characterisation of thin films with different number of layers using SCM. In addition, influence of pH and ionic strength on the fabrication results of thin films was studied. SCM allowed us to determine the film thickness at the single layer level and to understand the relationship between surface morphology and fabrication conditions. SCM can visualise the change in morphology as a function of the number of layers of thin films and measure thickness at the 3.5 nm/bilayer level.
In our laboratory of physics of living systems, tumour cells were studied using SCM, cytotoxic effects of cisplatin and nocodazole on morphological parameters of cells were evaluated [13], morphological changes of erythrocytes during transformation into echinocyte and acanthocyte were sdudied [14], the use of platinum electrodes for detection of reactive oxygen species was demonstrated [15], and human embryonic stem cells were visualised (Fig.1).
In addition to direct studies of biological objects, we are actively engaged in development of our own capillary microscopy platform. Our group managed to combine the advantages of SCM in a compact implementation of the FemtoScan Xi microscope (Fig.2) [16].
CONCLUSIONS
SCM offers many new advantages in the study of living cells. Three-dimensional morphology contributes to a better understanding of the process of osteogenic differentiation of mesenchymal stem cells and can be used as an important indicator in further clinical applications.
SCM allows imaging and measuring the mechanical properties of living platelets, which is important in the study of diseases associated with abnormal platelet cytoskeleton, such as uremia. Using a capillary, chemical or physical stimuli can be applied to the area of the membrane where cell vibrations are recorded.
SCM provides valuable information for studying the effects of drug-induced apoptosis in long-term experiments on living cells.
By combining SCMs with highly selective Pt-functionalised electrodes, real-time monitoring of extracellular and intracellular hydrogen peroxide concentrations became possible, providing valuable information on reactive oxygen species-mediated processes at the level of individual cells.
SCM can be used to optimise conditions for preparing of layer-by-layer films used for various purposes such as encapsulation of enzymes and functionalisation of molecules. SCM has been actively developed in recent years and finds wide applications in regenerative and biomedical applications, single cell research and other fields.
ACKNOWLEDGMENTS
The work of the staff of the Physical Department was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru). The image of stem cells was obtained by T.O.Sovetnikov.
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.
Scanning capillary microscopy (SCM) is another type of probe microscopy used to obtain three-dimensional topographic information of biological samples in liquid [1]. SCM measures the ionic current through a capillary filled with electrolyte, which strongly depends on the distance between the probe and the sample. The SCM resolution is at the level of nanometre units, slightly losing to atomic force microscopy (AFM), but the non-contact nature of SCM measurements does not damage biological samples, which are characterised by reduced mechanical stiffness. SCM makes it possible not only to obtain static pictures of cells, but also to carry out long-term monitoring of living biological objects without affecting the sample and to get important data on their biomechanical properties. Therefore, scanning capillary microscopy becomes an indispensable tool for study of living systems.
The membrane of living human erythrocytes carries out oscillations of submicron scale, which have been mainly studied by optical microscopy. The functional role of this phenomenon is still unclear; the membrane oscillations amplitude is regarded as an indicator of mechanical resistance to stresses occurring in capillary beds. It is not easy to experimentally measure membrane oscillations; the main challenge is to develop methods that allow tracking very small displacements at a very high rate, preferably over a large area and for a long time [2].
In [3], cell membrane oscillations dynamics in erythrocytes was studied by analysing the ionic current noise recorded near the cell membrane using SCM. A capillary with a radius of about 300 nm collected data on oscillations with an amplitude of about 200 nm at the cell membrane. Ion current noise was recorded at a fixed distance from the cell and used to determine the amplitude of the membrane oscillations. The slope of the current-distance curve was used as a conversion factor. It was found that the amplitude of oscillations decreased with increasing membrane tension caused by different methods. Decrease in osmolarity of the medium caused swelling of cells and decrease in the oscillations amplitude. A similar result was obtained when polylysine concentration was increased a hundredfold: this treatment increased cell adhesion and stiffness. Both cellular and local increases in membrane tension caused a decrease in the cell membrane oscillations amplitude.
Platelet activation plays a crucial role in haemostasis and thrombosis. It is well known that platelets control retraction and compaction of the blood clot, but their mechanical properties have rarely been studied. Scanning capillary microscopy has been used to visualise morphological and mechanical properties of living human platelets with high spatial resolution [4]. Their mean elastic modulus was presented to decrease during thrombin-induced activation by approximately twofold. A similar softening of platelets was observed during cytoskeleton depolymerisation induced by cytochalasin D, and the researchers were able to distinguish between the effects of thrombin and cytochalasin D.
Human mesenchymal stem cells (MSCs) are interesting as a research object for regenerative medicine in various diseases. Osteogenic differentiation is one of the capabilities of MSCs, which is crucial for their clinical applications. Cell deformability and changes in mechanical properties during cell death were recorded using AFM [5, 6]. During adipogenic and osteogenic differentiation [7], topography of MSCs, osteoblasts and osteosarcoma cells were studied using AFM [8]. The results showed that cell height may be an important factor in osteogenic differentiation. With the help of SCM in [9] we quantitatively assessed long-term changes in MSC topography during osteogenic differentiation. It was shown that different cell lines undergo different morphological changes during osteogenic differentiation.
In [10], 3D morphology and roughness of A549 adenocarcinoma cells under physiological conditions before and after cisplatin-induced apoptosis for 24 hours were studied. Tracing morphology of the same single A549 cells exposed to cisplatin revealed heterogeneity of response to the drug, formation of membrane bubbles and increased membrane roughness.
Colorectal cancer is closely related to the level of hydrogen peroxide (H2O2) in the tumour microenvironment. Several clinical studies on the use of H2O2 in cancer treatment have revealed its paradoxical role as a stimulator of cancer progression. A study [11] combined SCM with highly sensitive Pt-functionalised electrodes to measure dynamic extracellular and intracellular H2O2 gradients in individual Caco-2 colorectal cancer cells. The relationship between cell mechanical properties and H2O2 gradients was revealed. Exposure to H2O2 at 0.1 or 1 mmol/L concentration increased the gradient of extracellular and intracellular H2O2. Remarkably, cellular F-actin-dependent stiffness increased at 0.1 mmol/L but decreased when H2O2 eustresses 1 mmol/L. This study shows the complex interplay between physical properties and biochemical signals in the antioxidant defence of tumour cells, demonstrating the use of H2O2 eustress for survival at the level of individual cells. Inhibition of glutathione peroxidase 2 or catalase enhances the cytotoxic activity of H2O2 eustress against colorectal cancer cells, which holds promise for the development of H2O2 related therapies for cancer and other inflammatory diseases.
The layer-by-layer film deposition method is the alternate adsorption of anionic and cationic polyelectrolytes via electrostatic interactions. In order to prepare thin films with desired structure and properties, it is important to understand how different fabrication conditions affect the thin films formation. In [12], they performed in situ characterisation of thin films with different number of layers using SCM. In addition, influence of pH and ionic strength on the fabrication results of thin films was studied. SCM allowed us to determine the film thickness at the single layer level and to understand the relationship between surface morphology and fabrication conditions. SCM can visualise the change in morphology as a function of the number of layers of thin films and measure thickness at the 3.5 nm/bilayer level.
In our laboratory of physics of living systems, tumour cells were studied using SCM, cytotoxic effects of cisplatin and nocodazole on morphological parameters of cells were evaluated [13], morphological changes of erythrocytes during transformation into echinocyte and acanthocyte were sdudied [14], the use of platinum electrodes for detection of reactive oxygen species was demonstrated [15], and human embryonic stem cells were visualised (Fig.1).
In addition to direct studies of biological objects, we are actively engaged in development of our own capillary microscopy platform. Our group managed to combine the advantages of SCM in a compact implementation of the FemtoScan Xi microscope (Fig.2) [16].
CONCLUSIONS
SCM offers many new advantages in the study of living cells. Three-dimensional morphology contributes to a better understanding of the process of osteogenic differentiation of mesenchymal stem cells and can be used as an important indicator in further clinical applications.
SCM allows imaging and measuring the mechanical properties of living platelets, which is important in the study of diseases associated with abnormal platelet cytoskeleton, such as uremia. Using a capillary, chemical or physical stimuli can be applied to the area of the membrane where cell vibrations are recorded.
SCM provides valuable information for studying the effects of drug-induced apoptosis in long-term experiments on living cells.
By combining SCMs with highly selective Pt-functionalised electrodes, real-time monitoring of extracellular and intracellular hydrogen peroxide concentrations became possible, providing valuable information on reactive oxygen species-mediated processes at the level of individual cells.
SCM can be used to optimise conditions for preparing of layer-by-layer films used for various purposes such as encapsulation of enzymes and functionalisation of molecules. SCM has been actively developed in recent years and finds wide applications in regenerative and biomedical applications, single cell research and other fields.
ACKNOWLEDGMENTS
The work of the staff of the Physical Department was carried out under the state order with the financial support of the Physical Department of the Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by Advanced Technologies Center (www.nanoscopy.ru). The image of stem cells was obtained by T.O.Sovetnikov.
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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