Amyloid fibrils are primarily known in a pathogenic context for their association with a wide range of debilitating human diseases. Here we show a marine invertebrate (Entobdella soleae) utilizes functional amyloid fibrils comparable to those of a unicellular prokaryote (Escherichia coli). Thioflavin-T binding and Raman spectroscopy provided evidence for the presence of amyloid in the adhesive of Entobdella soleae. We elucidated that for these two very different organisms, amyloid fibrils provide adhesive and cohesive strength to their natural adhesives. Comparing the nanoscale mechanical responses of these fibrils with those of pathogenic amyloid by atomic force microscopy revealed that the molecular level origin of the cohesive strength was associated with the generic intermolecular β-sheet structure of amyloid fibrils. Functional adhesive residues were found only in the case of the functional amyloid. Atomic force microscopy provided a useful means to characterize the internal structural forces within individual amyloid fibrils and how these relate to the mechanical performance of both functional and pathogenic amyloid. The mechanistic link of amyloid-based cohesive and adhesive strength could be widespread amongst natural adhesives, irrespective of environment, providing a new strategy for biomimicry and a new source of materials for understanding the formation and stability of amyloid fibrils more generally.

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High-speed atomic force microscopy (HS AFM) in ‘contact' mode was used to image at video rate the surfaces of both calcium hydroxyapatite samples, often used as artificial dental enamel in such experiments, and polished actual bovine dental enamel in both neutral and acidic aqueous environments. The image in each frame of the video of the sample was a few micrometers square, and the high-speed scan window was panned across the sample in real time to examine larger areas. Conventional AFM images of the same regions of the sample were also recorded before and after high-speed imaging. The ability of HS AFM to follow processes occurring in liquid on the timescale of a few seconds was employed to study the dissolution process of both hydroxyapatite and bovine enamel under acidic conditions. Buffered citric acid at pH values between 3.0 and 4.0 was observed to dissolve the surface layers of these samples. The movies recorded showed rapid dissolution of the bovine enamel in particular, which proceeded until the relatively small amount of acid available had been exhausted. A comparison was made with enamel samples that had been treated in fluoride solution (1 h in 300 ppm NaF, pH 7) prior to addition of the acid; the speed of dissolution for these samples was much less than that of the untreated samples. The HS AFM used an in-house designed and constructed high-speed flexure scan stage employing a push-pull piezo actuator arrangement. The HS AFM is able to follow the large changes in height (on the micrometer scale) that occur during the dissolution process.

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Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC^TM (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image. In this study, we first demonstrated structural properties of purified α Actinin-4 by conventional AFM. Since this molecule is an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction, we investigated their structural properties using the recognition mode of AFM. For this purpose, we attached an anti-α Actinin-4 monoclonal antibody to the AFM cantilever and performed recognition imaging against α Actinin-4. We finally succeeded in mapping the epitopic region within the α Actinin-4 molecule. Thus, recognition imaging using an antibody coupled AFM cantilever will be useful for single-molecule anatomy of biological macromolecules and structures.

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The mechanical memory effect of single cells was reported in our recent study. In order to clarify this effect, various sequential stimuli of uniaxial deformation were applied to cells by deformable culture dishes and a deformation device, and the local stiffness distribution of single C2C12 myoblasts was visualized by scanning probe microscopy. Following a single step stretching, cellular stiffness first increased steeply and then gradually decreased for two hours. By a single step stretching 30 min after a long pulse-like deformation with a pulse duration of 30 min, the cells responded in the same way. On the other hand, they did not respond to a single step stretching 30 min after a short pulse-like deformation with a pulse duration of 0.5 min. These results indicated that cellular mechanical response to external deformation is affected strongly by a preceding deformation and that the duration time of the preceding deformation is an important factor in the change in mechanical response. We consider that the change in mechanical response contributes to a regulatory mechanism of cellular contractile force.

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Scanning probe microscopy (SPM) provides a range of strategies for studying biological phenomena due to its ability to image surfaces under liquids. However, some cellular events, such as cell migration, exceed the maximum measurable range of SPM. Recently, we have developed a wide range scanning probe microscope (WR-SPM) to investigate cellular events which exceed the range of the conventional SPM. In this review, we introduce the instrumentation of the WR-SPM, which can measure a sample for 400 μm in the xy directions and 23 μm in the z direction. We then show the application of the WR-SPM to studies of the stiffness response of epithelial cells to an external loading force and demonstrat that the stiffness of the epithelial cells increases under stretched conditions. We also showed the results on the mesh structure on the surface of a melanoma cell as well as the regulatory mechanism of the cellular contractile force by the combined use of topographical and mechanical modes of the WR-SPM. These findings indicate that the WR-SPM is very useful for studying the functions of a cell in relation to the surface structure and mechanical properties of that cell.

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The relationship between the higher-order structure of human metaphase chromosomes and the distribution of topoisomerase IIα was analyzed by a comparison of atomic force microscope (AFM) and fluorescence microscope images of the same chromosome. AFM imaging of chromosomes in liquid revealed the presence of alternating ridges and grooves on the surfaces of the sister chromatids. In contrast, the fluorescence image of the chromosomes stained with the anti-topoisomerase IIα antibody showed that the fluorescence intensity of topoisomerase IIα was not uniform and that there were alternating strong and weak spots along the chromosome axes. A comparison of the AFM image with a fluorescence microscope image of the same chromosome further demonstrated that ridges and grooves corresponded to strong and weak fluorescence intensities of topoisomerase IIα, respectively. These findings suggest that the distribution of topoisomerase IIα has a close connection with the higher-order structure of human metaphase chromosomes.

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We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E^* | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.

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We have been developing a low invasive cell manipulation technology based on inserting an ultra-thin needle--“nanoneedle”--into a living cell by using an atomic force microscope (AFM). The nanoneedle, made from a silicon AFM tip by focused-ion-beam etching, has a diameter of several hundred nanometers and a length of about 10 microns. Successful insertion of the nanoneedle into the cell can be confirmed by the appearance of a steep relaxation of repulsive force in the force-distance curve as monitored by the AFM system. This technology, termed “cell surgery”, can be applied for the detection of intracellular proteins in a living cell or for highly efficient gene transfer. The present study shows that the durability of a tapered nanoneedle is superior to that of a cylindrical nanoneedle, and that a proper aspect ratio for the tapered nanoneedle must be chosen to maintain sufficient insertion efficiency for a particular target cell: tapered nanoneedles of an aspect ratio over 20 showed high insertion efficiency for various kinds of mammalian cells. We then used diamond for the material of the nanoneedle because its specific properties, such as high stiffness, heat conductivity, and electrical conductivity capacitated by boron doping, were deemed useful for the analysis and manipulation of intracellular phenomena. We compared the capability of the diamond nanoneedle in cell manipulation with that of the silicon nanoneedle. Evaluation of the effect of the former on transcription efficiency and localization analysis of p53 expression revealed the low invasiveness for cell manipulation as was also the case for the silicon nanoneedle. We also succeeded in achieving highly efficient plasmid DNA delivery into a mouse fibroblast C3H10T1/2 using the diamond nanoneedle. The diamond nanoneedle is expected to contribute to the versatility of “cell surgery” technology.

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We developed a novel nano manipulator based on an atomic force microscope (AFM) that can be operated inside the sample chamber of a scanning electron microscope (SEM). This AFM manipulator is also coupled with a haptic device, and the nanometer-scale movement of the AFM cantilever can be scaled up to the millimeter-scale movement of the pen handle of the haptic device. Using this AFM manipulation system, we were able to observe the AFM cantilever and samples under the SEM and obtain topographical images of the AFM under the SEM. These AFM images contained quantitative height information of the sample that is difficult to obtain from SEM images. Our system was also useful for positioning the cantilever for accurate AFM manipulation because the manipulation scene could be directly observed in real time by SEM. Coupling of the AFM manipulator with the haptic device was also useful for manipulation in the SEM since the operator can move the AFM probe freely at any position on the sample surface while feeling the interaction force between the probe and the sample surface. We tested two types of cutting methods: simple cutting and vibration cutting. Our results showed that vibration cutting with probe oscillation is very useful for the dissection of biological samples which were dried for SEM observation. Thus, cultivated HeLa cells were successfully micro-dissected by vibration cutting, and the dissection process could be observed in real time in the SEM. This AFM manipulation system is expected to serve as a powerful tool for dissecting various biological samples at the micro and nanometer-scale under SEM observation.

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