AFM对小鼠胚胎干细胞的形态学研究

目的：对已建系BALB/c小鼠胚胎干细胞膜表面进行纳米级超微结构的初步形态学研究,从而为从分子水平研究胚胎干细胞的增殖与分化调控机理奠定基础。方法：利用原子力显微镜（AFM）,在空气中对小鼠胚胎干细胞膜表面扫描成像。结果：AFM图像表现BALB/c小鼠胚胎干细胞呈圆盘状,直径约10-15μm,高约2-4μm,胚胎干细胞膜表面比较复杂,随着扫描范围的减小,切向分辨率逐渐增大,可达到纳米分辨,细胞表面有许多紧密堆积的椭球状颗粒。颗粒尺寸（x-y方向）为40-80nm。结果：利用AFM可以得到胚胎干细胞表面高分辨的,可重复的图像。     

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.     

Atomic force microscopy and chemical force microscopy of microbial cells

Over the past years, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of microbial cells with nanometer resolution, and under physiological conditions. Moreover, chemical force microscopy (CFM) and single-molecule force spectroscopy have enabled researchers to map chemical groups and receptors on cell surfaces, providing valuable insight into their structure-function relationships. Here, we present protocols for analyzing spores of the pathogen Aspergillus fumigatus using real-time AFM imaging and CFM. We emphasize the use of porous polymer membranes for immobilizing single live cells, and the modification of gold-coated tips with alkanethiols for CFM measurements. We also discuss recording conditions and data interpretation, and provide recommendations for reliable experiments. For well-trained AFM users, the entire protocol can be completed in 2-3 d.     

Atomic force microscopy imaging of lipid rafts of human breast cancer cells

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.     

Atomic force microscopy imaging and pulling of nucleic acids

Recent advances in atomic force microscopy (AFM) imaging of nucleic acids include the visualization of DNA and RNA incorporated into devices and patterns, and into structures based on their sequences or sequence recognition. AFM imaging of nuclear structures has contributed to advances in telomere research and to our understanding of nucleosome formation. Highlights of force spectroscopy or pulling of nucleic acids include the use of DNA as a programmable force sensor, and the analysis of RNA flexibility and drug binding to DNA.     

Atomic force microscopy imaging of dried or living bacteria.

Atomic force microscopy (AFM) was used to obtain micrographs of dried bacteria in air, and of living ones in their culture medium. Images of dried bacteria were very similar to images obtained elsewhere by the much more complicated cryoetching preparation technique for transmission electron microscopy. Living bacteria were immobilized on a poly-L-lysine film, and directly observed in their culture medium at a resolution unattainable by any other technique applicable to living material. The images were similar to those obtained in scanning electron microscopy where the specimen must be fixed, dried and coated with conductive material, and as a result, no longer viable.     

Atomic force microscopy imaging of DNA under macromolecular crowding conditions

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl 2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt.     

Atomic force microscopy imaging and mechanical properties measurement of red blood cells and aggressive cancer cells

Mechanical properties play an important role in regulating cellular activities and are critical for unlocking the mysteries of life. Atomic force microscopy (AFM) enables researchers to measure mechanical properties of single living cells under physiological conditions. Here, AFM was used to investigate the topography and mechanical properties of red blood cells (RBCs) and three types of aggressive cancer cells (Burkitt??s lymphoma Raji, cutaneous lymphoma Hut, and chronic myeloid leukemia K562). The surface topography of the RBCs and the three cancer cells was mapped with a conventional AFM probe, while mechanical properties were investigated with a micro-sphere glued onto a tip-less cantilever. The diameters of RBCs are significantly smaller than those of the cancer cells, and mechanical measurements indicated that Young??s modulus of RBCs is smaller than those of the cancer cells. Aggressive cancer cells have a lower Young??s modulus than that of indolent cancer cells, which may improve our understanding of metastasis.     

Atomic force microscopy: from cell imaging to molecular manipulation

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angstr?ms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.     

Atomic force microscopy: from cellular imaging to molecular manipulation

Using a sharp tip attached at the end of a soft cantilever as a probe, the atomic force microscope (AFM) explores the surface topography of biological samples bathed in physiological solutions. In the last few years, the AFM has gained popularity among biologists. This has been obtained through the improvement of the equipment and imaging techniques as well as through the development of new non-imaging applications. Biological imaging has to face a main difficulty that is the softness and the dynamics of most biological materials. Progress in understanding the AFM tip-biological samples interactions provided spectacular results in different biological fields. Recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules at work are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra- and intermolecular forces from single molecules are also presented.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Atomic force microscopy imaging reveals the domain structure of polycystin-1

Mutation of polycystin-1 (PC1) is the major cause of autosomal dominant polycystic kidney disease. PC1 has a predicted molecular mass of ~460 kDa comprising a long multidomain extracellular N-terminal region, 11 transmembrane regions, and a short C-terminal region. Because of its size, PC1 has proven difficult to handle biochemically, and structural information is consequently sparse. Here we have isolated wild-type PC1, and several mutants, from transfected cells by immunoaffinity chromatography and visualized individual molecules using atomic force microscopy (AFM) imaging. Full-length PC1 appeared as two unequally sized blobs connected by a 35 nm string. The relative sizes of the two blobs suggested that the smaller one represents the N-terminus, including the leucine-rich repeats, the first polycystic kidney disease (PKD) domain, and the C-type lectin motif, while the larger one is the C-terminus, including the receptor for egg jelly (REJ) domain, all transmembrane domains, and the cytoplasmic tail. The intervening string would then consist of a series of tandem PKD domains. The structures of the various PC1 mutants were all consistent with this model. Our results represent the first direct visualization of the structure of PC1, and reveal the architecture of the protein, with intriguing implications for its function.     

Atomic force microscopy and proteins

This review briefly introduces the principles of atomic force microscopy (AFM) applied to protein samples. AFM provides three-dimensional surface images of the proteins with high resolution. The advantage of AFM for protein studies is that AFM can visualize directly the molecule under physiological conditions without previous treatment. AFM operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand receptor interactions with the goal of measuring forces at the individual molecule level.     

Atomic force microscopy of animal cells: Advances and prospects

We review the advances of the method of atomic force microscopy (AFM) for investigating the animal cells and analyze its development, paying much attention to studies of living cells. We consider the specific features and tasks of AFM, and a number of special AFM-based techniques. We discuss the choice of probe geometry for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and analysis of the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches, as well as combining AFM with optical and laser scanning confocal microscopy, and the possible applications of AFM in biotechnology and medicine.     

Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa

Atomic force microscopy (AFM) has emerged as the only technique capable of real-time imaging of the surface of a living cell at nano-resolution. Since AFM provides the advantage of directly observing living biological cells in their native environment, this technique has found many applications in pharmacology, biotechnology, microbiology, structural and molecular biology, genetics and other biology-related fields. AFM has also proved to be a valuable tool for reproductive biologists. An exhaustive review on the various applications of AFM to sperm cells is presented. AFM has been extensively applied for determining the structural and topological features of spermatozoa. Unstained, unfixed spermatozoa in their natural physiological surroundings can be imaged by this technique which provides valuable information about the morphological and pathological defects in sperm cells as three-dimensional images with precise topographical details. Sperm head defects and the acrosome at the tip of the head responsible for fertilization, can be examined and correlated with the lack of functional integrity of the cell. Considerable amount of work is reported on the structural details of the highly condensed chromatin in sperm head using AFM. Detailed information on 3D topographical images of spermatozoa acquired by AFM is expected to provide a better understanding of various reproductive pathways which, in turn, can facilitate improved infertility management and/or contraceptive development.     

Atomic force microscopy comes of age

AFM (atomic force microscopy) analysis, both of fixed cells, and live cells in physiological environments, is set to offer a step change in the research of cellular function. With the ability to map cell topography and morphology, provide structural details of surface proteins and their expression patterns and to detect pico‐Newton force interactions, AFM represents an exciting addition to the arsenal of the cell biologist. With the explosion of new applications, and the advent of combined instrumentation such as AFM—confocal systems, the biological application of AFM has come of age. The use of AFM in the area of biomedical research has been proposed for some time, and is one where a significant impact could be made. Fixed cell analysis provides qualitative and quantitative subcellular and surface data capable of revealing new biomarkers in medical pathologies. Image height and contrast, surface roughness, fractal, volume and force analysis provide a platform for the multiparameter analysis of cell and protein functions. Here, we review the current status of AFM in the field and discuss the important contribution AFM is poised to make in the understanding of biological systems.