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18 pages, 4926 KiB

Open AccessArticle

Red Blood Cell Storage with Xenon: Safe or Disruption?

by Ekaterina Sherstyukova, Viktoria Sergunova, Snezhanna Kandrashina, Aleksandr Chernysh, Vladimir Inozemtsev, Galina Lomakina and Elena Kozlova

Cells 2024, 13(5), 411; https://doi.org/10.3390/cells13050411 - 27 Feb 2024

Viewed by 1050

Abstract

Xenon, an inert gas commonly used in medicine, has been considered as a potential option for prolonged preservation of donor packed red blood cells (pRBCs) under hypoxic conditions. This study aimed to investigate how xenon affects erythrocyte parameters under prolonged storage. In vitro [...] Read more.

Xenon, an inert gas commonly used in medicine, has been considered as a potential option for prolonged preservation of donor packed red blood cells (pRBCs) under hypoxic conditions. This study aimed to investigate how xenon affects erythrocyte parameters under prolonged storage. In vitro model experiments were performed using two methods to create hypoxic conditions. In the first method, xenon was introduced into bags of pRBCs which were then stored for 42 days, while in the second method, xenon was added to samples in glass tubes. The results of our experiment showed that the presence of xenon resulted in notable alterations in erythrocyte morphology, similar to those observed under standard storage conditions. For pRBC bags, hemolysis during storage with xenon exceeded the acceptable limit by a factor of six, whereas the closed-glass-tube experiment showed minimal hemolysis in samples exposed to xenon. Notably, the production of deoxyhemoglobin was specific to xenon exposure in both cell suspension and hemolysate. However, this study did not provide evidence for the purported protective properties of xenon. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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14 pages, 11506 KiB

Open AccessArticle

Cell Surface Parameters for Accessing Neutrophil Activation Level with Atomic Force Microscopy

by Oksana M. Tilinova, Vladimir Inozemtsev, Ekaterina Sherstyukova, Snezhanna Kandrashina, Mikhail Pisarev, Andrey Grechko, Nina Vorobjeva, Viktoria Sergunova and Maxim E. Dokukin

Cells 2024, 13(4), 306; https://doi.org/10.3390/cells13040306 - 7 Feb 2024

Cited by 2 | Viewed by 1221

Abstract

In this study, we examine the topography and adhesion images of the cell surface of neutrophils during the activation process. Our analysis of cell surface parameters indicates that the most significant changes in neutrophils occur within the first 30 min of activation, suggesting [...] Read more.

In this study, we examine the topography and adhesion images of the cell surface of neutrophils during the activation process. Our analysis of cell surface parameters indicates that the most significant changes in neutrophils occur within the first 30 min of activation, suggesting that reactive oxygen species may require approximately this amount of time to activate the cells. Interestingly, we observed surface granular structure as early as 10 min after neutrophil activation when examining atomic force microscopy images. This finding aligns with the reorganization observed within the cells under confocal laser scanning microscopy. By analyzing the cell surface images of adhesion, we identified three spatial surface parameters that correlate with the activation time. This finding enables us to estimate the degree of activation by using atomic force microscopy maps of the cell surface. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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21 pages, 23317 KiB

Open AccessArticle

Correlation of Plasma Membrane Microviscosity and Cell Stiffness Revealed via Fluorescence-Lifetime Imaging and Atomic Force Microscopy

by Yuri M. Efremov, Liubov Shimolina, Alexander Gulin, Nadezhda Ignatova, Margarita Gubina, Marina K. Kuimova, Peter S. Timashev and Marina V. Shirmanova

Cells 2023, 12(21), 2583; https://doi.org/10.3390/cells12212583 - 6 Nov 2023

Cited by 2 | Viewed by 2491

Abstract

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of [...] Read more.

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells’ stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young’s modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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11 pages, 1238 KiB

Open AccessFeature PaperArticle

Machine Learning Allows for Distinguishing Precancerous and Cancerous Human Epithelial Cervical Cells Using High-Resolution AFM Imaging of Adhesion Maps

by Mikhail Petrov and Igor Sokolov

Cells 2023, 12(21), 2536; https://doi.org/10.3390/cells12212536 - 28 Oct 2023

Cited by 1 | Viewed by 1443

Abstract

Previously, the analysis of atomic force microscopy (AFM) images allowed us to distinguish normal from cancerous/precancerous human epithelial cervical cells using only the fractal dimension parameter. High-resolution maps of adhesion between the AFM probe and the cell surface were used in that study. [...] Read more.

Previously, the analysis of atomic force microscopy (AFM) images allowed us to distinguish normal from cancerous/precancerous human epithelial cervical cells using only the fractal dimension parameter. High-resolution maps of adhesion between the AFM probe and the cell surface were used in that study. However, the separation of cancerous and precancerous cells was rather poor (the area under the curve (AUC) was only 0.79, whereas the accuracy, sensitivity, and specificity were 74%, 58%, and 84%, respectively). At the same time, the separation between premalignant and malignant cells is the most significant from a clinical point of view. Here, we show that the introduction of machine learning methods for the analysis of adhesion maps allows us to distinguish precancerous and cancerous cervical cells with rather good precision (AUC, accuracy, sensitivity, and specificity are 0.93, 83%, 92%, and 78%, respectively). Substantial improvement in sensitivity is significant because of the unmet need in clinical practice to improve the screening of cervical cancer (a relatively low specificity can be compensated by combining this approach with other currently existing screening methods). The random forest decision tree algorithm was utilized in this study. The analysis was carried out using the data of six precancerous primary cell lines and six cancerous primary cell lines, each derived from different humans. The robustness of the classification was verified using K-fold cross-validation (K = 500). The results are statistically significant at p < 0.0001. Statistical significance was determined using the random shuffle method as a control. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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14 pages, 3002 KiB

Open AccessArticle

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM

by Sergey Dubkov, Aleksei Overchenko, Denis Novikov, Vasilii Kolmogorov, Lidiya Volkova, Petr Gorelkin, Alexander Erofeev and Yuri Parkhomenko

Cells 2023, 12(21), 2521; https://doi.org/10.3390/cells12212521 - 25 Oct 2023

Cited by 1 | Viewed by 1506

Abstract

The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for [...] Read more.

The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum–thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young’s modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope–spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 10⁵. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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15 pages, 3413 KiB

Open AccessArticle

Measuring Melanoma Nanomechanical Properties in Relation to Metastatic Ability and Anti-Cancer Drug Treatment Using Scanning Ion Conductance Microscopy

by Emily Woodcock, Peter V. Gorelkin, Philip S. Goff, Christopher R. W. Edwards, Yanjun Zhang, Yuri Korchev and Elena V. Sviderskaya

Cells 2023, 12(19), 2401; https://doi.org/10.3390/cells12192401 - 4 Oct 2023

Cited by 2 | Viewed by 1841

Abstract

A cell’s mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, reliable cancer marker. For example, it has been widely published that cancer cells show a lower Young’s modulus than their non-cancerous [...] Read more.

A cell’s mechanical properties have been linked to cancer development, motility and metastasis and are therefore an attractive target as a universal, reliable cancer marker. For example, it has been widely published that cancer cells show a lower Young’s modulus than their non-cancerous counterparts. Furthermore, the effect of anti-cancer drugs on cellular mechanics may offer a new insight into secondary mechanisms of action and drug efficiency. Scanning ion conductance microscopy (SICM) offers a nanoscale resolution, non-contact method of nanomechanical data acquisition. In this study, we used SICM to measure the nanomechanical properties of melanoma cell lines from different stages with increasing metastatic ability. Young’s modulus changes following treatment with the anti-cancer drugs paclitaxel, cisplatin and dacarbazine were also measured, offering a novel perspective through the use of continuous scan mode SICM. We found that Young’s modulus was inversely correlated to metastatic ability in melanoma cell lines from radial growth, vertical growth and metastatic phases. However, Young’s modulus was found to be highly variable between cells and cell lines. For example, the highly metastatic cell line A375M was found to have a significantly higher Young’s modulus, and this was attributed to a higher level of F-actin. Furthermore, our data following nanomechanical changes after 24 hour anti-cancer drug treatment showed that paclitaxel and cisplatin treatment significantly increased Young’s modulus, attributed to an increase in microtubules. Treatment with dacarbazine saw a decrease in Young’s modulus with a significantly lower F-actin corrected total cell fluorescence. Our data offer a new perspective on nanomechanical changes following drug treatment, which may be an overlooked effect. This work also highlights variations in cell nanomechanical properties between previous studies, cancer cell lines and cancer types and questions the usefulness of using nanomechanics as a diagnostic or prognostic tool. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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20 pages, 4582 KiB

Open AccessArticle

Mechanical Properties and Nanomotion of BT-20 and ZR-75 Breast Cancer Cells Studied by Atomic Force Microscopy and Optical Nanomotion Detection Method

by Maria N. Starodubtseva, Nastassia M. Shkliarava, Irina A. Chelnokova, María I. Villalba, Andrei Yu. Krylov, Eldar A. Nadyrov and Sandor Kasas

Cells 2023, 12(19), 2362; https://doi.org/10.3390/cells12192362 - 26 Sep 2023

Cited by 1 | Viewed by 1592

Abstract

Cells of two molecular genetic types of breast cancer—hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)—were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we [...] Read more.

Cells of two molecular genetic types of breast cancer—hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)—were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young’s modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200–300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young’s modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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18 pages, 14244 KiB

Open AccessArticle

Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study

by Viktoria Sergunova, Vladimir Inozemtsev, Nina Vorobjeva, Elena Kozlova, Ekaterina Sherstyukova, Snezhanna Lyapunova and Aleksandr Chernysh

Cells 2023, 12(17), 2199; https://doi.org/10.3390/cells12172199 - 2 Sep 2023

Cited by 4 | Viewed by 2230

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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11 pages, 3156 KiB

Open AccessCommunication

Exploring the Process of Neutrophil Transendothelial Migration Using Scanning Ion-Conductance Microscopy

by Svetlana N. Pleskova, Nikolay A. Bezrukov, Ekaterina N. Gorshkova, Sergey Z. Bobyk and Ekaterina V. Lazarenko

Cells 2023, 12(13), 1806; https://doi.org/10.3390/cells12131806 - 7 Jul 2023

Cited by 2 | Viewed by 1672

Abstract

The dynamics of neutrophil transendothelial migration was investigated in a model of experimental septicopyemia. Scanning ion-conductance microscopy allowed us to determine changes in morphometric characteristics of endothelial cells during this process. In the presence of a pyogenic lesion simulated by Staphylococcus aureus, [...] Read more.

The dynamics of neutrophil transendothelial migration was investigated in a model of experimental septicopyemia. Scanning ion-conductance microscopy allowed us to determine changes in morphometric characteristics of endothelial cells during this process. In the presence of a pyogenic lesion simulated by Staphylococcus aureus, such migration was accompanied by both compensatory reactions and alteration of both neutrophils and endothelial cells. Neutrophils demonstrated crawling along the contact sites between endothelial cells, swarming phenomenon, as well as anergy and formation of neutrophil extracellular traps (NETs) as a normergic state. Neutrophil swarming was accompanied by an increase in the intercellular spaces between endothelial cells. Endothelial cells decreased the area of adhesion to the substrate, which was determined by a decrease in the cell projection area, and the cell membrane was smoothed. However, endothelial cell rigidity was paradoxically unchanged compared to the control. Over time, neutrophil migration led to a more significant alteration of endothelial cells: first, shallow perforations in the membrane were formed, which were repaired rather quickly, then stress fibrils were formed, and finally, endothelial cells died and multiple perforations were formed on their membrane. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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13 pages, 2797 KiB

Open AccessArticle

Investigation of the Antifungal and Anticancer Effects of the Novel Synthesized Thiazolidinedione by Ion-Conductance Microscopy

by Nikita Savin, Alexander Erofeev, Roman Timoshenko, Alexander Vaneev, Anastasiia Garanina, Sergey Salikhov, Natalia Grammatikova, Igor Levshin, Yuri Korchev and Petr Gorelkin

Cells 2023, 12(12), 1666; https://doi.org/10.3390/cells12121666 - 19 Jun 2023

Cited by 2 | Viewed by 1534

Abstract

In connection with the emergence of new pathogenic strains of Candida, the search for more effective antifungal drugs becomes a challenge. Part of the preclinical trials of such drugs can be carried out using the innovative ion-conductance microscopy (ICM) method, whose unique [...] Read more.

In connection with the emergence of new pathogenic strains of Candida, the search for more effective antifungal drugs becomes a challenge. Part of the preclinical trials of such drugs can be carried out using the innovative ion-conductance microscopy (ICM) method, whose unique characteristics make it possible to study the biophysical characteristics of biological objects with high accuracy and low invasiveness. We conducted a study of a novel synthesized thiazolidinedione’s antimicrobial (for Candida spp.) and anticancer properties (on samples of the human prostate cell line PC3), and its drug toxicity (on a sample of the human kidney cell line HEK293). We used a scanning ion-conductance microscope (SICM) to obtain the topography and mechanical properties of cells and an amperometric method using Pt-nanoelectrodes to register reactive oxygen species (ROS) expression. All data and results are obtained and presented for the first time. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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Review

Jump to: Research

16 pages, 3420 KiB

Open AccessReview

Scanning Probe Microscopy Techniques for Studying the Cell Glycocalyx

by Dmitry Kolesov, Anna Astakhova, Maria Galdobina, Alexey Moskovtsev, Aslan Kubatiev, Alisa Sokolovskaya, Leonid Ukrainskiy and Sergey Morozov

Cells 2023, 12(24), 2778; https://doi.org/10.3390/cells12242778 - 6 Dec 2023

Cited by 1 | Viewed by 1230

Abstract

The glycocalyx is a brush-like layer that covers the surfaces of the membranes of most cell types. It consists of a mixture of carbohydrates, mainly glycoproteins and proteoglycans. Due to its structure and sensitivity to environmental conditions, it represents a complicated object to [...] Read more.

The glycocalyx is a brush-like layer that covers the surfaces of the membranes of most cell types. It consists of a mixture of carbohydrates, mainly glycoproteins and proteoglycans. Due to its structure and sensitivity to environmental conditions, it represents a complicated object to investigate. Here, we review studies of the glycocalyx conducted using scanning probe microscopy approaches. This includes imaging techniques as well as the measurement of nanomechanical properties. The nanomechanics of the glycocalyx is particularly important since it is widely present on the surfaces of mechanosensitive cells such as endothelial cells. An overview of problems with the interpretation of indirect data via the use of analytical models is presented. Special insight is given into changes in glycocalyx properties during pathological processes. The biological background and alternative research methods are briefly covered. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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23 pages, 13446 KiB

Open AccessReview

Analytical Models for Measuring the Mechanical Properties of Yeast

by Nikita Savin, Alexander Erofeev and Petr Gorelkin

Cells 2023, 12(15), 1946; https://doi.org/10.3390/cells12151946 - 27 Jul 2023

Cited by 2 | Viewed by 1488

Abstract

The mechanical properties of yeast play an important role in many biological processes, such as cell division and growth, maintenance of internal pressure, and biofilm formation. In addition, the mechanical properties of cells can indicate the degree of damage caused by antifungal drugs, [...] Read more.

The mechanical properties of yeast play an important role in many biological processes, such as cell division and growth, maintenance of internal pressure, and biofilm formation. In addition, the mechanical properties of cells can indicate the degree of damage caused by antifungal drugs, as the mechanical parameters of healthy and damaged cells are different. Over the past decades, atomic force microscopy (AFM) and micromanipulation have become the most widely used methods for evaluating the mechanical characteristics of microorganisms. In this case, the reliability of such an estimate depends on the choice of mathematical model. This review presents various analytical models developed in recent years for studying the mechanical properties of both cells and their individual structures. The main provisions of the applied approaches are described along with their limitations and advantages. Attention is paid to the innovative method of low-invasive nanomechanical mapping with scanning ion-conductance microscopy (SICM), which is currently starting to be successfully used in the discovery of novel drugs acting on the yeast cell wall and plasma membrane. Full article

(This article belongs to the Special Issue Advances in Scanning Probe Microscopy in Cell Biology)

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