Structural biology has solved the atomic structure of many proteins, but all revealed are static snapshots. Single-molecule biophysics has recorded the dynamic action of proteins but only in an indirect way of observing optical markers attached to the molecules. To overcome these limitations, various techniques were developed towards a great speed performance of AFM. Through these efforts, high-speed AFM (HS-AFM) was established a decade ago. HS-AFM enabled for the first time direct observation of protein molecules during their functional activity. A few tens of different protein systems have already been observed with HS-AFM. These imaging studies have demonstrated that HS-AFM can provide deep insights into the functional mechanism of proteins inaccessible with other approaches. This line of imaging studies are now actively carried out. In parallel with this activity, further technical developments have also been performed towards the functional extension of HS-AFM. The targets of HS-AFM is now being expanded from purified molecules to organelles, bacteria, and eukaryotic cells. This review first describes the outline of HS-AFM techniques and its application studies performed so far. Then, it shows remaining technical issues to be solved and proposes ways to overcome these issues.
Progresses in microscopic techniques have ever contributed to many discoveries in biology. Electron microscopic techniques have revealed that cell membrane structures are organized into functional domains, whereas optical microscopic techniques such as fluorescence imaging have revealed the dynamics of specific molecules in live cell membrane. However, neither technique can directly and simultaneously visualize the structure and dynamics of living cell surface at high spatiotemporal resolution.
High speed atomic force microscopy (HS-AFM) is powerful tool which visualize the dynamic molecular process in physiological condition. This technique has ever been succeeded in imaging many kinds of proteins purified from cell at high spatiotemporal resolution. However, there are not many applications for cell imaging. In this study, we applied this technique for observing bacterial cell surface. AFM images showed nanostructure and the dynamic processes on bacterial cell outer membrane. Here, we report the result of direct observation on live cell surface using HS-AFM.
Mycoplasma mobile, a fish pathogenic bacterium lacking a peptidoglycan layer glides on a host cell surface with a unique mechanism. In the gliding mechanism, the force is generated through ATP hydrolysis on a gliding motor, evolved from F-type ATPase. We visualized the structural changes of gliding motors in living cells by using high-speed atomic force microscopy (high-speed AFM) which is a unique method to directly observe the dynamic molecules. The high-speed AFM visualized particle structures forming chains at intervals about 30 nm in the cell, consistent with the gliding motor observed by electron microscopy. Half of particles showed 8 nm shift to the left side relative to the gliding direction of cell and 5 nm sink to the cytoplasm side. The others stayed static through the observation. The frequency of the movement was reduced by sodium azide, F-type ATPase inhibitor, suggesting that the movement depends on ATP hydrolysis. In the gliding mechanism, the movement should be converted and amplified to the step of “leg” through “crank”, respectively composed of Gli349 and Gli521 on cell surface.
Propolis is a resin-like natural product produced by mixing saliva with sap and pollen by bees and is also used in folk medicine for periodontal treatment. In this study, we examined the antimicrobial activity of propolis ethanol extract against Porphyromonas gingivalis, which is regarded as a keystone among periodontopathic bacteria. As a result, it was found that P. gingivalis showed high sensitivity to propolis, and propolis showed a bactericidal effect more effective than ampicillin. In addition, we observed non-immobilized live P. gingivalis cells using high-speed atomic force microscopy system BIXAM, and succeeded in observing in real-time how propolis induces abnormal membrane vesicle-like structures on the cell surface. These findings suggest that propolis has excellent selective antibacterial activity against P. gingivalis, and suggest its applicability to the prevention and treatment of periodontal disease.
Recent developments and applications of aberration-corrected DPC STEM with segmented detector are reviewed. DPC STEM can directly image internal structures of individual atoms by observing atomic electric field as demonstrated by imaging single Au atoms and single layer graphene. Total charge density mapping for GaN crystal using DPC STEM is highlighted, showing direct imaging of electron cloud surrounding atomic nuclei. Some practical applications such as observing pn junction in semiconductors and magnetic skyrmion in helimagmets are shown.
Cilia are slender projections with a length of 1 to 10 μm and a diameter of about 200 nm, and are evolutionarily conserved very well from protozoans such as Paramecium to vertebrates including humans. In multicellular organisms such as vertebrates, a primary cilium protrudes on a cell, and in mammals almost all cells in the body possess it. The primary cilium is a fine structure as thin as the optical limit of light microscopy. It is easily overlooked as only a tiny structure is present on the surface of cells with a size of several tens of μm, while those who know the existence can find it out through careful observation. In addition, primary cilia are an unstable structure compared to many motile cilia of Paramecium and tracheal epithelium. The fragility of primary cilia makes findings false negative, i.e. loss of cilia, or false positive, i.e. abnormal structures, as artifacts unless the samples are handled carefully. I herein introduce difficulties and some tips of observing such “fragile” primary cilia, and show what are observed with an optical microscope—the morphology and behavior of primary cilia.
Cryo-EM revolutionary advancement enables us to solve biological macromolecular structures up to 1.5 Å spatial resolution. This brings up new issues, which had not received much attention before. Coma and cold field emission (CFE) are representative examples of these issues. This article describes how and why coma affects cryo-EM imaging and what should be done for collection of high-quality data. Coma introduces phase shifts, which becomes larger in higher resolution and more problematic for accurate structure determination. The effect should be carefully minimized. The merit of CFE is reviewed for higher-resolution single particle cryo-EM. The CFE source produces an electron beam with high temporal-coherence, and yields contrast transfer function curves to be remarkably less attenuated at a resolution range beyond 2 Å than those from the standard thermal field emission or Schottky source. Thus, this technology should drive cryo-EM to the next stage. Many new researchers have now started using cryo-EM and I think that the imaging theory for high-resolution cryo-EM should be updated to be fitted with these situations. I describe this article for this purpose. It also includes our recent single particle reconstructions obtained with a CRYO ARM 300 electron microscope equipped with a CFE gun.
A novel double-slit experiment was performed using the state-of-the-art electron wave interference technology. Using an asymmetric double-slit, we conducted experiments called “which-way” experiments to determine which slit electrons passed through under the condition of a short propagation distance named as a pre-Fraunhofer condition. Individual electrons were categorized into three types; those that passed though only the left slit; those that passed though both slits simultaneously; and those that passed through only the right slit. The distribution of those electrons was explicitly shown in color-coded figure.