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.     

Purification and pH stability characterization of a chymotrypsin inhibitor from Schizolobium parahyba seeds

Schizolobium parahyba chymotrypsin inhibitor (SPCI) was completely purified as a single polypeptide chain with two disulfide bonds, by TCA precipitation and ion exchange chromatography. This purification method is faster and more efficient than that previously reported: SPCI is stable from pH 2 to 12 at 25 degrees C, and is highly specific for chymotrypsin at pH 7-12. It weakly inhibits elastase and has no significant inhibitory effect against trypsin and alpha-amylase. SPCI is a thermostable protein and resists thermolysin digestion up to 70 degrees C.     

pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds

The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.     

Effects of denaturing and stabilizing agents on the inhibitory activity and conformational stability of Schizolobium parahyba chymotrypsin inhibitor

The conformational stability of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) was investigated based on conformational changes and inhibitory activity in the presence of chaotropic and stabilizing agents. At 90°C, the half-lifetime of SPCI was 154 min, while in the presence of 1 M KCl and 20% PEG 20,000, it was drastically reduced to 6 and 3 min, respectively. In contrast, at 90°C, the SPCI structure remained unaltered with the addition of 1 mM DTT and 56% glycerol. The reduction of the two disulfide bonds caused conformational changes in the SPCI without altering the inhibitory activity, suggesting that disulfide bonds are irrelevant to the maintenance of SPCI conformation. Unfolded structures were formed in the presence of 6 M GdnHCl, while in the presence of 8 M urea, destabilization was due to peptide bond rupture. These results suggest that the thermal inactivation of SPCI involves conformational changes and that hydrophobic and electrostatic interactions play a significant role, while the disulfide bonds are of secondary importance in maintaining the high thermal stability of SPCI.     

Analysis of ligand-receptor interactions in cells by atomic force microscopy

Atomic force microscopy (AFM) increasingly has been used to analyse "receptor" function, either by using purified proteins ("molecular recognition microscopy") or, more recently, in situ in living cells. The latter approach has been enabled by the use of a modified commercial AFM, linked to a confocal microscope, which has allowed adhesion forces between ligands and receptors in cells to be measured and mapped, and downstream cellular responses analysed. We review the application of AFM to cell biology and, in particular, to the study of ligand-receptor interactions and draw examples from our own work and that of others to show the utility of AFM, including for the exploration of cell surface functionalities. We also identify shortcomings of AFM in comparison to "standard" methods, such as receptor auto-radiography or immuno-detection, that are widely applied in cell biology and pharmacological analysis.     

Conformational dynamics of individual antibodies using computational docking and AFM

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen‐binding fragments and one crystallizable fragment using a surface‐constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X‐ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X‐ray structure consequence of the antibody flexibility. We conclude that high‐resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica. Copyright © 2013 John Wiley & Sons, Ltd.     

不同厚度细胞薄切片对 AFM 图像反差的影响

利用原子力显微镜（ AFM ）观察超薄切片的表面,探索表面形貌与切片厚度、朝向等因素的关系以及对图像反差的影响 . 选择三种不同类型的细胞,培养后按电镜超薄切片法固定、包埋并切片后,将不同厚度的切片区分上下表面转移到云母上, AFM 在空气中以接触模式进行观察 . 结果发现,切片表面细胞相对包埋介质的凸起与凹陷与切片本身的厚度密切相关,并随切片厚度的不同呈现有规律的变化 . 实验统计结果显示这种现象可能具有普遍性 .     

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.     

Simultaneous confidence intervals for pairwise multiple comparisons in a two-way unbalanced design

Turkey's (1953, The Problem of Multiple Comparisons, unpublished report, Princeton University) procedure is widely used for pairwise multiple comparisons in one-way ANOVA. It provides exact simultaneous pairwise confidence intervals (SPCI) for balanced designs and conservative SPCI for unbalanced designs. In this paper, we will extend Turkey's procedure to two-way unbalanced designs. Both the exact and the conservative methods will be introduced. The application of the new procedure is illustrated with sample data from two experiments.     

Tracking Molecular Interactions in Membranes by Simultaneous ATR-FTIR-AFM

In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.     

Red blood cells imaging and antigen-antibody interaction measurement

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.     

How to orient the functional GroEL-SR1 mutant for atomic force microscopy investigations

We present high-resolution atomic force microscopy (AFM) imaging of the single-ring mutant of the chaperonin GroEL (SR-EL) from Escherichia coli in buffer solution. The native GroEL is generally unsuitable for AFM scanning as it is easily being bisected by forces exerted by the AFM tip. The single-ring mutant of GroEL with its simplified composition, but unaltered capability of binding substrates and the co-chaperone GroES, is a more suited system for AFM studies. We worked out a scheme to systematically investigate both the apical and the equatorial faces of SR-EL, as it binds in a preferred orientation to hydrophilic mica and hydrophobic highly ordered pyrolytic graphite. High-resolution topographical imaging and the interaction of the co-chaperone GroES were used to assign the orientations of SR-EL in comparison with the physically bisected GroEL. The usage of SR-EL facilitates single molecule studies on the folding cycle of the GroE system using AFM.     

Myelinating and demyelinating phenotype of Trembler-J mouse (a model of Charcot-Marie-Tooth human disease) analyzed by atomic force microscopy and confocal microscopy

The accumulation of misfolded proteins is associated with various neurodegenerative conditions. Mutations in PMP-22 are associated with the human peripheral neuropathy, Charcot-Marie-Tooth Type 1A (CMT1A). PMP-22 is a short-lived 22 kDa glycoprotein, which plays a key role in the maintenance of myelin structure and compaction, highly expressed by Schwann cells. It forms aggregates when the proteasome is inhibited or the protein is mutated. This study reports the application of atomic force microscopy (AFM) as a detector of profound topographical and mechanical changes in Trembler-J mouse (CMT1A animal model). AFM images showed topographical differences in the extracellular matrix and basal lamina organization of Tr-J/+ nerve fibers. The immunocytochemical analysis indicated that PMP-22 protein is associated with type IV collagen (a basal lamina ubiquitous component) in the Tr-J/+ Schwann cell perinuclear region. Changes in mechanical properties of single myelinating Tr-J/+ nerve fibers were investigated, and alterations in cellular stiffness were found. These results might be associated with F-actin cytoskeleton organization in Tr-J/+ nerve fibers. AFM nanoscale imaging focused on topography and mechanical properties of peripheral nerve fibers might provide new insights into the study of peripheral nervous system diseases.     

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.     

Ultrastructural studies on the collagen of the marine sponge Chondrosia reniformis Nardo

The ultrastructure of isolated fibrils of Chondrosia reniformis sponge collagen was investigated by collecting characteristic data, such as fibril thickness, width, D-band periodicity, and height modulation, using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Therefore an adapted pre-processing of the insoluble collagen into homogeneous suspensions using neutral buffer solutions was essential, and several purification steps have been developed. Fourier transform infrared reflection-absorption spectroscopy (FT-IRAS) of the purified sponge collagen showed remarkable analogy of peak positions and intensities with the spectra of fibrillar calf skin type I collagen, despite the diverse phylogenetic and evolutionary origin. The sponge collagen's morphology is compared with that of other fibrillar collagens, and the typical banding of the separated single fibrils is discussed by comparison of topographical data obtained using AFM and corresponding TEM investigations using common staining methods. As the TEM images of the negatively stained fibrils showed alternating dark and light bands, AFM revealed a characteristic periodicity of protrusions (overlap zones) followed by two equal interband regions (gap zones). AFM and TEM results were correlated and multiperiodicity in Chondrosia collagen's banding is demonstrated. The periodic dark bands observed in TEM images correspond directly to the periodic protrusions seen by AFM. As a result, we provide an improved, updated model of the collagen's structure and organization.     

Direct visualization of polypeptide shell of ferritin molecule by atomic force microscopy.

The polypeptide shell of the ferritin molecule has been imaged in water by atomic force microscopy (AFM). The central dip and the quaternary structure could be observed on the surface of the ferritin molecule anchored inhomogeneously in two dimensions. These structures observed in the AFM images are quite similar to the electron density map near the top of the apoferritin viewed down from a 4-fold axis structure reported previously (S. H. Banyard, D. K. Stammers, and P. M. Harrison, 1978. Nature (Lond.). 271:282-284). It has been achieved by introducing a "self-screening effect" of the surface charges of the AFM sample (S. Ohnishi, M. Hara, T. Furuno, and H. Sasabe. 1992. Biophys. J. 63:1425-1431) and the specially sharpened stylus of AFM cantilever.     

Simultaneous topography and recognition imaging on endothelial cells

Determining the landscape of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometres) is an important task in many fields of biological science. During the past five years, dynamic recognition imaging (e.g. simultaneous topography and recognition (TREC) imaging) has proven to be a powerful technique in biophysical research. This technique becomes an indispensable tool for high-resolution receptor mapping as it has been successfully demonstrated on different biomolecular model systems. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a flexible and distensible tether. In this review, we describe the principles of TREC imaging and provide a flavour of its recent application on endothelial cells.     

Structure of intracellular mature vaccinia virus visualized by in situ atomic force microscopy

Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended approximately 16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.     

Parallel AFM imaging and force spectroscopy using two-dimensional probe arrays for applications in cell biology

Atomic force microscopy (AFM) investigations of living cells provide new information in both biology and medicine. However, slow cell dynamics and the need for statistically significant sample sizes mean that data collection can be an extremely lengthy process. We address this problem by parallelizing AFM experiments using a two-dimensional cantilever array, instead of a single cantilever. We have developed an instrument able to operate a two-dimensional cantilever array, to perform topographical and mechanical investigations in both air and liquid. Deflection readout for all cantilevers of the probe array is performed in parallel and online by interferometry. Probe arrays were microfabricated in silicon nitride. Proof-of-concept has been demonstrated by analyzing the topography of hard surfaces and fixed cells in parallel, and by performing parallel force spectroscopy on living cells. These results open new research opportunities in cell biology by measuring the adhesion and elastic properties of a large number of cells. Both properties are essential parameters for research in metastatic cancer development.