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: 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 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: A powerful molecular toolkit in nanoproteomics

Analysing microbial cell surface proteins is a challenging task in current microbial proteomic research, which has major implications for drug design, vaccine development, and microbial monitoring. In this context, atomic force microscopy (AFM) has recently emerged has a powerful characterization platform, providing valuable insights into the surface proteome of microbial cells. The aim of this article is to show how advanced AFM techniques, that all have in common functionalization of the AFM tip with specific molecules, can be used to answer pertinent questions related to surface‐associated proteins, such as what is their spatial arrangement on the cell surface, and what are the forces driving their interaction with the environment?     

Atomic force microscopy imaging of live mammalian cells

Atomic force microscopy (AFM) was used to examine the morphology of live mammalian adherent and suspended cells. Time-lapse AFM was used to record the locomotion dynamics of MCF-7 and Neuro-2a cells. When a MCF-7 cell retracted, many small sawtooth-like filopodia formed and reorganized, and the thickness of cellular lamellipodium increased as the retraction progressed. In elongated Neuro-2a cells, the cytoskeleton reorganized from an irregular to a parallel, linear morphology. Suspended mammalian cells were immobilized by method combining polydimethylsiloxane-fabricated wells with poly-L-lysine electrostatic adsorption. In this way, the morphology of a single live lymphoma cell was imaged by AFM. The experimental results can improve our understanding of cell locomotion and may lead to improved immobilization strategies.     

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 studies on cellular elastic and viscoelastic properties

In this work, a method based on atomic force microscopy (AFM) approach-reside-retract experiments was established to simultaneously quantify the elastic and viscoelastic properties of single cells. First, the elastic and viscoelastic properties of normal breast cells and cancerous breast cells were measured, showing significant differences in Young’s modulus and relaxation times between normal and cancerous breast cells. Remarkable differences in cellular topography between normal and cancerous breast cells were also revealed by AFM imaging. Next, the elastic and viscoelasitc properties of three other types of cell lines and primary normal B lymphocytes were measured; results demonstrated the potential of cellular viscoelastic properties in complementing cellular Young’s modulus for discerning different states of cells. This research provides a novel way to quantify the mechanical properties of cells by AFM, which allows investigation of the biomechanical behaviors of single cells from multiple aspects.     

Atomic force microscopy investigation of vaccinia virus structure

Vaccinia virus was treated in a controlled manner with various combinations of nonionic detergents, reducing agents, and proteolytic enzymes, and successive products of the reactions were visualized using atomic force microscopy (AFM). Following removal of the outer lipid/protein membrane, a layer 20 to 40 nm in thickness was encountered that was composed of fibrous elements which, under reducing conditions, rapidly decomposed into individual monomers on the substrate. Beneath this layer was the virus core and its prominent lateral bodies, which could be dissociated or degraded with proteases. The core, in addition to the lateral bodies, was composed of a thick, multilayered shell of proteins of diverse sizes and shapes. The shell, which was readily etched with proteases, was thoroughly permeated with pores, or channels. Prolonged exposure to proteases and reductants produced disgorgement of the viral DNA from the remainders of the cores and also left residual, flattened, protease-resistant sacs on the imaging substrate. The DNA was readily visualized by AFM, which revealed some regions to be “soldered” by proteins, others to be heavily complexed with protein, and yet other parts to apparently exist as bundled, naked DNA. Prolonged exposure to proteases deproteinized the DNA, leaving masses of extended, free DNA. Estimates of the interior core volume suggest moderate but not extreme compaction of the genome.     

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 study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze-etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.     

Atomic force microscope,molecular imaging,and analysis

Image visibility is a central issue in analyzing all kinds of microscopic images. An increase of intensity contrast helps to raise the image visibility, thereby to reveal fine image features. Accordingly, a proper evaluation of results with current imaging parameters can be used for feedback on future imaging experiments. In this work, we have applied the Laplacian function of image intensity as either an additive component (Laplacian mask) or a multiplying factor (Laplacian weight) for enhancing image contrast of high‐resolution AFM images of two molecular systems, an unknown protein imaged in air, provided by AFM COST Action TD1002 ( http://www.afm4nanomedbio.eu /), and tobacco mosaic virus (TMV) particles imaged in liquid. Based on both visual inspection and quantitative representation of contrast measurements, we found that the Laplacian weight is more effective than the Laplacian mask for the unknown protein, whereas for the TMV system the strengthened Laplacian mask is superior to the Laplacian weight. The present results indicate that a mathematical function, as exemplified by the Laplacian function, may yield varied processing effects with different operations. To interpret the diversity of molecular structure and topology in images, an explicit expression for processing procedures should be included in scientific reports alongside instrumental setups. Copyright © 2015 John Wiley & Sons, Ltd.     

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 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 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 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 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 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 in biology: technology and techniques

In this review, I describe the biological applications of the atomic force microscope (AFM). The historical background and the development of the microscope are described. The AFM can operate in many different modes relevant to biological systems including topography, chemical analysis, and forces relevant at the biological length scale (single cell to DNA dimensions and pico to nano Newton forces). A limited number of examples from the literature are described to illustrate some of the many capabilities of this microscope. The aim is to give an introduction of the technique to the inexperienced in this rapidly growing field.     

Atomic force microscopy in biology: technology and techniques

In this review, I describe the biological applications of the atomic force microscope (AFM). The historical background and the development of the microscope are described. The AFM can operate in many different modes relevant to biological systems including topography, chemical analysis, and forces relevant at the biological length scale (single cell to DNA dimensions and pico to nano Newton forces). A limited number of examples from the literature are described to illustrate some of the many capabilities of this microscope. The aim is to give an introduction of the technique to the inexperienced in this rapidly growing field.