Observation of living cells using the atomic force microscope.

We used an atomic force microscope (AFM) to image samples immersed in a fluid in order to study the dynamic behavior of the membranes of living cells. AFM images of cultured cells immersed in a buffer were obtained without any preliminary preparation. We observed surface changes and displacements which suggest that the cells were still alive during the measurements. Some membrane details imaged with the AFM have also been observed using a scanning electron microscope and their dynamic behavior has been confirmed by microcinematography. We believe that the AFM will offer new insights into the exploration of dynamic changes affecting cell membranes.     

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

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 microm 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 approximately 5 and 30 nm xy size.     

Imaging cells with the atomic force microscope

Different types of cells have been imaged with the atomic force microscope. The morphology of the archaebacterium Halobacterium halobium in its dry state was revealed. On a leaf of the small Indian tree Lagerstroemia subcostata a stoma was imaged. The lower side of a water lily leaf was imaged in water showing features down to 12 nm. Finally, fixed red and white blood cells were imaged in buffer showing features down to 8 nm. The images demonstrate that atomic force microscopy can provide high-resolution images of cell surfaces under physiological conditions.     

Gadolinium induces domain and pore formation of human erythrocyte membrane: an atomic force microscopic study.

Lanthanide cations bind to human erythrocyte membranes and enhance cell permeability. It was postulated that this effect is due to their likeness with calcium ions, which have been used to induce perforation of cells. However, the nature and mechanism of the perforation are still not clear. In the present work, the change in surface topography of erythrocyte membranes exposed to various gadolinium species was imaged with an atomic force microscope (AFM) in order to get direct evidence of perforation. The images of the whole cell and regions in nanometer scale showed that the normal surface is featured by closely packed nanometer size particles. The AFM images showed that Gd(3+) binding to erythrocytes led to domain structure at low concentration and pore formation at higher concentration. The domain structures that appeared after incubation with 1.0x10(-6)-1.0x10(-5) mol/l Gd(3+) solution for 30 min are featured by the particles aggregated to form ranges and the separations among them enlarged to gorges. With a higher concentration, 2.5x10(-5) mol/l Gd(3+), the further aggregation developed into crater-shaped 'pores'. By washing with EDTA the 'pores' can be resealed but the domain structure remained. The anionic complex of Gd(3+), [Gd(Cit)(2)](3-) of this concentration, can only induce the domain structure formation. The domain and 'pore' structures mediated by Gd(3+) concentrations might be responsible for both enhanced permeability and perforation. The mechanism of Gd-induced domain formation and perforation is discussed on the basis of aggregation of membrane proteins and the coexistence of different phases of membrane lipids resulting from Gd(3+) binding.     

Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy.

The redistribution of platelet constituents during activation is essential for their physiological function of maintaining hemostasis. We report here about real time investigations of the activation of native human platelets under physiological conditions from the initial formation of filopodia to the fully spread form by atomic force microscopy. We followed the trafficking of granules and their interaction with the plasma membrane within single cells. Our results show movement of certain granula towards the lamellipodia. Analysis of this rearrangement and the subsequent enlargement of the platelet surface reveals details of the membrane spreading process. Images of living cells are presented that show the distribution of cytoskeletal components and membrane-bound filaments at a resolution of better than 50 nm. The local minimum forces between the tip and the platelets were estimated to be smaller than 60 pN. A model for the elastic contributions of the glycocalix to the tip/membrane interaction was developed using the theory of grafted polymers.     

Extraction of membrane proteins from a living cell surface using the atomic force microscope and covalent crosslinkers

The force curve mode of the atomic force microscope (AFM) was applied to extract intrinsic membrane proteins from the surface of live cells using AFM tips modified by amino reactive bifunctional covalent crosslinkers. The modified AFM tips were individually brought into brief contact with the living cell surface to form covalent bonds with cell surface molecules. The force curves recorded during the detachment process from the cell surface were often characterized by an extension of a few hundred nanometers followed mostly by a single step jump to the zero force level. Collection and analysis of the final rupture force revealed that the most frequent force values (of the force) were in the range of 0.4–0.6 nN. The observed rupture force most likely represented extraction events of intrinsic membrane proteins from the cell membrane because the rupture force of a covalent crosslinking system was expected to be significantly larger than 1.0 nN, and the separation force of noncovalent ligand-receptor pairs to be less than 0.2 nN, under similar experimental conditions. The transfer of cell surface proteins to the AFM tip was verified by recording characteristic force curves of protein stretching between the AFM tips used on the cell surface and a silicon surface modified with amino reactive bifunctional crosslinkers. This method will be a useful addition to bionanotechnological research for the application of AFM.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

Microscopic assessment of membrane protein structure and function

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.     

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.     

AN ATOMIC FORCE MICROSCOPIC EXAMINATION OF THE APICAL MEMBRANE OF THE MAMMALIAN GASTRIC GLAND

The authors have examined the morphology of the apical membrane of living gastric glands from both the rat and rabbit with an atomic force microscope using both a conventional upright configuration and in the new inverted bioscope mode. Individual gastric glands were hand dissected and the apical membrane was exposed using a microsurgical approach. The split open glands allowed to access and directly image 4–6 cells with their apical axis available to the scanning tip of the atomic force microscope. All cells were scanned in a physiological Ringer solution at 37°C. The scans revealed that we could visualize both parietal and chief cells and to distinguish them via their unique apical topography. The parietal cells showed a variety of small canaliculi that were dispersed along the apical axis of the cell. Scans of chief cells revealed an apical surface that was covered with microvilli along the entire apical margin. The results of these studies show that it is indeed feasible to image living gastric glands at 37°C and to observe the surface topology of both the parietal and chief cell.     

Progress in the analysis of membrane protein structure and function

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.     

Effect of Cold Plasma on Glial Cell Morphology Studied by Atomic Force Microscopy

The atomic force microscope (AFM) is broadly used to study the morphology of cells. The morphological characteristics and differences of the cell membrane between normal human astrocytes and glial tumor cells are not well explored. Following treatment with cold atmospheric plasma, evaluation of the selective effect of plasma on cell viability of tumor cells is poorly understood and requires further evaluation. Using AFM we imaged morphology of glial cells before and after cold atmospheric plasma treatment. To look more closely at the effect of plasma on cell membrane, high resolution imaging was used. We report the differences between normal human astrocytes and human glioblastoma cells by considering the membrane surface details. Our data, obtained for the first time on these cells using atomic force microscopy, argue for an architectural feature on the cell membrane, i.e. brush layers, different in normal human astrocytes as compared to glioblastoma cells. The brush layer disappears from the cell membrane surface of normal E6/E7 cells and is maintained in the glioblastoma U87 cells after plasma treatment.     

Topological Structures and Membrane Nanostructures of Erythrocytes after Splenectomy in Hereditary Spherocytosis Patients via Atomic Force Microscopy

Hereditary spherocytosis is an inherited red blood cell membrane disorder resulting from mutations of genes encoding erythrocyte membrane and cytoskeletal proteins. Few equipments can observe the structural characteristics of hereditary spherocytosis directly expect for atomic force microscopy In our study, we proved atomic force microscopy is a powerful and sensitive instrument to describe the characteristics of hereditary spherocytosis. Erythrocytes from hereditary spherocytosis patients were small spheroidal, lacking a well-organized lattice on the cell membrane, with smaller cell surface particles and had reduced valley to peak distance and average cell membrane roughness vs. those from healthy individuals. These observations indicated defects in the certain cell membrane structural proteins such as α- and β-spectrin, ankyrin, etc. Until now, splenectomy is still the most effective treatment for symptoms relief for hereditary spherocytosis. In this study, we further solved the mysteries of membrane nanostructure changes of erythrocytes before and after splenectomy in hereditary spherocytosis by atomic force microscopy. After splenectomy, the cells were larger, but still spheroidal-shaped. The membrane ultrastructure was disorganized and characterized by a reduced surface particle size and lower than normal Ra values. These observations indicated that although splenectomy can effectively relieve the symptoms of hereditary spherocytosis, it has little effect on correction of cytoskeletal membrane defects of hereditary spherocytosis. We concluded that atomic force microscopy is a powerful tool to investigate the pathophysiological mechanisms of hereditary spherocytosis and to monitor treatment efficacy in clinical practices. To the best of our knowledge, this is the first report to study hereditary spherocytosis with atomic force microscopy and offers important mechanistic insight into the underlying role of splenectomy.     

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8±0.09 nm in diameter and 1.9±0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40―60 nm, and a range of 1.8―5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12―40 nm for their diameter and 0.7―2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 μm2. According to their size, we classified the invaginated pits into two types―larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead pro-toplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still de-tectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in di-ameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6―9.8 nm, thicker in regions covered with proteins.     

Structure of the extracellular surface of the gap junction by atomic force microscopy.

The extracellular surface of the gap junction cell-to-cell channels was imaged in phosphate-buffered saline with an atomic force microscope. The fully hydrated isolated gap junction membranes adsorbed to mica were irregular sheets approximately 1-2 microns across and 13.2 (+/- 1.3) nm thick. The top bilayer of the gap junction was dissected by increasing the force applied to the tip or sometimes by increasing the scan rate at moderate forces. The exposed extracellular surface revealed a hexagonal array with a center-to-center spacing of 9.4 (+/- 0.9) nm between individual channels (connexons). Images of individual connexons with a lateral resolution of < 3.5 nm, and in the best case approximately 2.5 nm, were reliably and reproducibly obtained with high-quality tips. These membrane channels protruded 1.4 (+/- 0.4) nm from the extracellular surface of the lipid membrane, and the atomic force microscope tip reached up to 0.7 nm into the pore, which opened up to a diameter of 3.8 (+/- 0.6) nm on the extracellular side.     

Visualization of the structures of the hepatitis C virus replication complex

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.     

A method for anchoring round shaped cells for atomic force microscope imaging.

More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM). However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers. This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape. Here we propose a simple and inexpensive solution to overcome these limitations. The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell. The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.     

Influence of synapsin I on synaptic vesicles: an analysis by force-volume mode of the atomic force microscope and dynamic light scattering

Synaptic vesicles (SVs) are small neuronal organelles that store neurotransmitters and release them by exocytosis into the synaptic cleft for signal transmission between nerve cells. They consist of a highly curved membrane composed of different lipids containing several proteins with specific functions. A family of abundant extrinsic SV proteins, the synapsins, interact with SV proteins and phospholipids and play an important role in the regulation of SV trafficking and stability. We investigated the interactions of one these proteins with the SV membrane using atomic force microscope and dynamic light scattering. We examined SVs isolated from rat forebrain both under native conditions and after depletion of endogenous synapsin I. We used the atomic force microscope in two modes: imaging mode for characterizing the shape and size of SVs, and force-volume mode for characterizing their stiffness. Synapsin-depleted SVs were larger in size and showed a higher tendency to aggregate than native vesicles, although their stiffness was not significantly different. Because synapsins are believed to cross-link SV to each other and to the actin cytoskeleton, we also measured the SV aggregation kinetics induced by synapsin I by dynamic light scattering and atomic force microscopy and found that the addition of synapsin I promotes a rapid aggregation of SVs. The data indicate that synapsin directly affects SV stability and aggregation state and support the physiological role of synapsins in the assembly and regulation of SV pools within nerve terminals.     

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.     

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to he a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.