Sample preparation and imaging of erythrocyte cytoskeleton with the atomic force microscopy (original) (raw)

Imaging erythrocytes under physiological conditions by atomic force microscopy

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2001

Since its invention in the mid 1980s atomic force microscopy has revolutionised the way in which surfaces can be imaged. Close to atomic resolution has been achieved for some materials and numerous images of molecules on surfaces have been recorded. Atomic force microscopy has also been of benefit to biology where protein molecules on surfaces have been studied and even whole cells have been investigated. Here we report a study of red blood cells which have been imaged in a physiological medium. At high resolution, the underlying cytoskeleton of the blood cell has been resolved and flaws in the cytoskeleton structure may be observed. Comparison of the normal`doughnut' shaped cells with swollen cells has been undertaken. Differences in both the global properties of the cells and in the local features in cytoskeleton structure have been observed. ß

Imaging the surface details of red blood cells with atomic force microscopy

Surface and Interface Analysis, 2002

ABSTRACT Atomic force microscopy (AFM) has been used in situ to study the topography and mechanical properties of red blood cells. When imaging the surface of the blood cells in topographic mode, the underlying cytoskeleton of the cell could be observed. This is a result of the different compressibility of the cell, depending on whether the underlying cytoskeleton is under the tip, which would be relatively hard, or just the cell membrane, which would be soft. The modulation technique confirms that the places where the cytoskeleton is directly underneath the membrane are raised as a result of the harder character and therefore the smaller tip indentation. The results obtained for the normal doughnut-shaped and swollen cells have been compared. Copyright © 2002 John Wiley & Sons, Ltd.

Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy

Journal of Biochemical and Biophysical Methods, 2006

During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-l-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes 0165-022X/$ -see front matter D was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases. D

Atomic force microscopy: From red blood cells to immunohaematology

Advances in Colloid and Interface Science, 2017

Atomic force microscopy (AFM) offers complementary imaging modes that can provide morphological and structural details of red blood cells (RBCs), and characterize interactions between specific biomolecules and RBC surface antigen. This review describes the applications of AFM in determining RBC health by the observation of cell morphology, elasticity and surface roughness. Measurement of interaction forces between plasma proteins and antibodies against RBC surface antigen using the AFM also brought new information to the immunohaematology field. With constant improvisation of the AFM in resolution and imaging time, the reaction of RBC to changes in the physico-chemistry of its environment and the presence of RBC surface antigen specific-biomolecules is achievable.

Imaging of the cytoplasmic leaflet of the plasma membrane by atomic force microscopy

PubMed, 1995

The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on glass coverslips was imaged by atomic force microscopy (AFM) in air and under aqueous medium, in "contact" mode. Micrometer range scans on air-dried samples revealed a heterogeneous structure with some filaments, likely corresponding to actin filaments that abut the inner leaflet of the membrane, and a few semi-organized lattice structures that might correspond to clathrin lattices. Experiments in phosphate-buffered saline confirmed the heterogeneity of the inner membrane surface with the presence of large (> 100 nm) globular structures emerging from the surface. Using sub-micrometer scan ranges, protruding particles, that occupy most of the membrane surface, were imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, high magnification images of the inner membrane surface were heterogeneous with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proximity.

Calcium-dependent human erythrocyte cytoskeleton stability analysis through atomic force microscopy

Journal of Structural Biology, 2005

Erythrocytes aVected by age and diseases such as sickle cell anemia, hypertension, diabetes, etc., exhibit abnormally high intracellular Ca 2+ ion levels, and appear to have altered cytoskeleton properties. It has been proposed that extra binding of Ca 2+ to membrane-associated calmodulin attenuates the spectrin-ankyrin-Band 3 tether of the cytoskeleton to the cytoplasmic membrane and might change the cytoskeleton structure. Due to the close apposition of the network, direct observation of such a structural change in vivo is restricted. In this study, atomic force microscopy and quantitative image analysis were applied to investigate the structural change of young healthy erythrocyte cytoskeletons upon extra Ca 2+ binding to the cytoplasmic membrane in vitro. The results show that extra Ca 2+ binding increased the cytoskeleton rigidity and prevented spectrin aggregation during sample preparation. The cytoskeleton morphology observed in Ca 2+ -incubated healthy young cell were similar to the glutaraldehyde-Wxed healthy young cells.

Imaging of the Surface of Living Cells by Low-Force Contact-Mode Atomic Force Microscopy

Biophysical Journal, 1998

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20 -50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20 -5 m scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between ϳ5 and 30 nm xy size.

Atomic Force Microscopy as a Tool for the Investigation of Living Cells

Medicina, 2013

Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low ...

Imaging of the Membrane Surface of MDCK Cells by Atomic Force Microscopy

The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions , including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.