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.     

Atomic force microscopy images suggest aggregation mechanism in cerato-platanin

Cerato-platanin (CP), the first member of the "cerato-platanin family", is a moderately hydrophobic protein produced by Ceratocystis fimbriata, the causal agent of a severe plant disease called "canker stain". The protein is localized in the cell wall of the fungus and it seems to be involved in the host-plane interaction and induces both cell necrosis and phytoalexin synthesis (one of the first plant defence-related events). Recently, it has been determined that CP, like other fungal surface protein, is able to self assemble in vitro. In this paper we characterize the aggregates of CP by Atomic Force Microscopy (AFM) images. We observe that CP tends to form early annular-shaped oligomers that seem to constitute the fundamental bricks of a hierarchical aggregation process, eventually resulting in large macrofibrillar assemblies. A simple model, based on the hypothesis that the aggregation is energetically favourable when the exposed surface is reduced, is compatible with the measured aggregates' shape and size. The proposed model can help to understand the mechanism by which CP and many other fungal surface proteins exert their effects.     

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.     

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.     

A complementary microscopy analysis of Sticholysin II crystals on lipid films: Atomic force and transmission electron characterizations

A new crystal form of the cytotoxin Sticholysin II (StnII) formed on lipid monolayers of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) has been characterized by transmission electron microscopy (TEM) and by tapping mode atomic force microscopy (AFM) under nearly physiological conditions. Both approaches show the existence of single- and double-layered 2D crystals possessing hexagonal symmetry and unit cell dimensions of a = b =10 nm and gamma = 120 degrees. However, single-layered StnII crystals could only be analysed by TEM and double-layered crystals by AFM. Considering the previously known atomic structure of native StnII and that of a tetrameric assembly, a model is proposed for this new crystal form in which StnII conserves its monomeric state upon interaction with the lipid monolayer. These results are in agreement with the existence of the so called M2 state of the actinoporins.     

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L_W = 174 nm and a number average contour length of L_N = 132 nm (L_W/L_N = 1.32). A proportion of the pectin molecules (30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L_W = 137 nm, L_N = 108 nm with L_W/L_N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.     

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 of Mammalian urothelial surface

The mammalian urothelium apical surface plays important roles in bladder physiology and diseases, and it provides a unique morphology for ultrastructural studies. Atomic force microscopy (AFM) is an emerging tool for studying the architecture and dynamic properties of biomolecular structures under near-physiological conditions. However, AFM imaging of soft tissues remains a challenge because of the lack of efficient methods for sample stabilization. Using a porous nitrocellulose membrane as the support, we were able to immobilize large pieces of soft mouse bladder tissue, thus enabling us to carry out the first AFM investigation of the mouse urothelial surface. The submicrometer-resolution AFM images revealed many details of the surface features, including the geometry of the urothelial plaques that cover the entire surface and the membrane interdigitation at the cell borders. This interdigitation creates a membrane zipper, likely contributing to the barrier function of the urothelium. In addition, we were able to image the intracellular bacterial communities of type 1-fimbriated bacteria grown between the intermediate filament bundles of the umbrella cells, shedding light on the bacterial colonization of the urothelium.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

女贞和珊瑚树叶片表面特征的AFM观察

应用原子力显微镜观察了女贞(Ligustrum lucidum)、珊瑚树(Viburnum odoratissimum)幼叶和成熟叶的表面特征,并探讨了叶面微结构对滞尘能力的可能影响以及抵抗干旱、污染物等胁迫的能力。女贞幼叶和成熟叶正背面的粗糙度Ra分别为417.8、794.5,1069、957.4 nm;珊瑚树幼叶和成熟叶正背面的粗糙度Ra分别为471.3、469.6,291.1、865.9 nm。和幼叶相比,成熟叶表面的粗糙度发生变化,但2个物种的变化趋势不同,这种变化可能与气孔的发育以及外界环境条件对叶片表面形态结构、蜡质含量和成分的影响不同有关。叶片表面存在大量的沟状、孔状峰谷区域和直径约为10 μm的凹陷,有利于PM₁₀的滞留。女贞和珊瑚树成熟叶气孔只分布在叶下表皮且下陷。这些特征均说明女贞和珊瑚树具有较强的滞尘能力和抵抗干旱、污染物胁迫的能力,作为绿篱植物对消减城市大气颗粒物污染和提高空气质量具有重要的意义。     

Changes in the stiffness of human mesenchymal stem cells with the progress of cell death as measured by atomic force microscopy

This note reports observations of the change of stiffness of human mesenchymal stem cells (hMSCs) with the progress of cell death as measured by AFM. hMSC with impaired membrane, dead and viable cells were labelled with Annexin V and Propidium Iodide after 24 h cold storage, followed by AFM measurement and Young's modulus of cells was derived. Viable hMSCs have a Young's modulus (E) in the range of 0.81–1.13 kPa and consistent measurement was observed when different measurement locations were chosen. E of cells with partially impaired membrane was 0.69±0.17 kPa or in the range of 2.04–4.74 kPa, depending upon the measurement locations. With the loss of membrane integrity, though there was no variation on measured E between different locations, a mixed picture of cell stiffness was observed as indicated by cells with E as low as 0.09±0.03 kPa, in a mid-range of 4.62±0.67 kPa, and the highest of up to 48.98±19.80 kPa. With the progress of cell death, the highest stiffness was noticed for cells showing a more granular appearance; also the lowest stiffness for cells with vacuole appearance. Findings from this study indicate that cell stiffness is significantly altered with the progress of cell death.     

Atomic force microscopy and cytochemistry of chromatin from marsupial spermatozoa with special reference to Sminthopsis crassicaudata

Atomic force microscopy (AFM) of the nuclear topology of spermatozoa from two marsupial species, Sminthopsis crassicaudata and Trichosurus vulpecula was investigated to determine the structural organisation of the chromatin subunits. That of the former species is of special interest as it has a peripheral nucleohistone region (C2) as well as a nuclease-resistant, nucleoprotamine core region (C1). Atomic force microscopy showed that the C2 region contained clusters of 120–160 nm nodules, whereas the C1 region exhibited smaller 50–80 nm nodules. The spermatozoa nuclei of Trichosurus, which has mainly nucleoprotamines, contained higher order chromatin structures of similar size to those from the C1 region of Sminthopsis. This study shows that nucleohistones and nucleoprotamines of marsupial sperm form distinct higher order conformations. For the second part of this work, the chromatin density and affinity for cationic stains of Sminthopsis spermatozoa were determined. Spermatozoa were observed with the transmission electron microscopy (TEM) either unstained or stained with metal salts. In the unstained specimens, the C2 nucleohistone region appeared more electron-lucent than the C1 region. When large cations such as uranyl were used, the reverse situation was observed. Therefore, the electron-dense appearance of the C2 chromatin in conventionally stained material may be due to the presence of net negative DNA charges that attract the cations used for EM staining, whereas the C1 chromatin may lack excess DNA negative charges that attract these stains and thus appears less electron-dense. Mol. Reprod. Dev. 48:367–374, 1997. © 1997 Wiley-Liss, Inc.     

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 μm, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for β-1,4-glucan and β-1,3-glucan.     

Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.     

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near‐native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4‐M23) was expressed in the X. laevis oocytes following their injection with AQP4‐M23 cRNA. AQP4‐M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4‐M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over‐expressed AQP4‐M23, the membranes from AQP4‐M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher‐order arrays of AQP4‐M23. In addition, but only infrequently, AQP4‐M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.