For the observation of moving objects, the image acquisition time should be short enough to avoid motion blur. However, most super-resolution fluorescent microscope techniques achieved high spatial resolution at the expense of temporal resolution, which precluded their application to live cell imaging. Here, we discuss the principle of the super-resolution microscopy based on the confocal microscope optics, which enabled us to observe the dynamics of subcellular organelles in living cells at the temporal resolution of 10 ms (100 frames per sec) and the spatial resolution around 100 nm (twice better than the diffraction limit).
Multi-photon excitation laser scanning fluorescence microscopy (MPLSM) has been widely used as an analytical method for direct visualization of dynamical molecular and cellular phenomena. This is because of its superior penetration depth and less invasiveness in specimens owing to its near-infrared excitation laser wavelength compared with the wavelength of single-photon excitation based systems. On the other hand, the spatial resolution with MPLSM tends to be inferior, mainly due to the wavelength of the excitation laser light. In this article, our spatial and temporal resolution improvements of MPLSM by utilizing novel optical technologies, such as the super-resolution microscopy or the spinning disk confocal microscopy, are described.
Super-resolution microscopy enables fluorescence imaging of biological molecules or structures at the spatial resolution of the molecular scale beyond the diffraction limit of light, and is promised to become essential for the research of molecular biology. However, the performance of super-resolution cannot be always achieved, especially in case of deep imaging inside living tissues. This problem is caused by the disturbance of light used for the imaging. When light is transmitted through living cells and tissues, which contains various structures with different refractive indices, the light is intricately disturbed. This caused the image degradation in conventional (not super-resolution) live-cell imaging, and the degradation becomes severer in super-resolution imaging, which involves precise arrangement of light or complex calculation. Adaptive optics (AO), the technique to correct the optical disturbance, started to be applied to live-cell imaging to perform high-resolution imaging inside living tissues. AO has been developed in astronomy to correct the disturbance of light caused by atmospheric turbulence, and is now indispensable for the high-resolution imaging using ground-based telescopes. Here, we introduce the overview of AO and researches to apply AO to live-cell and super-resolution imaging. We also discuss the future advancement of the AO research for super-resolution imaging.
SPoD-ExPAN is a super-resolution technique that uses a fluorescence microscope with illumination of patterned polarization and a reconstruction calculation of super-resolved images. In this technique the whole field from the microscope is acquired by a camera device at fast frame rates of 1–10 frames/s, and the spatial resolution is better than 100 nm. Many of conventional super-resolution microscopy techniques use illumination lights at extremely high power densities of 0.1 kW/cm2–1 GW/cm2, which unfortunately leads to serious phototoxicity to live cells and thereby difficulty in their time-lapse super-resolution observation. Here we have developed a novel super-resolution imaging technique using our recently-developed photoswithcable fluorescent protein Kohinoor and SPoD-ExPAN so that super-resolution observation can be achieved at an illumination power density of as low as ~1 W/cm2. This article presents the outline of the principle and the implementation of SPoD-ExPAN microscopy as well as the practical example of SPoD-ExPAN imaging using Kohinoor that we have performed.
The method for detecting magnetic circular (chiral) dichroic signals using energy-loss spectroscopy of fast electrons running through a sample is called as ‘Energy-loss magnetic chiral dichroism (EMCD) measurement’, which enables us to measure the magnetic moments localized in magnetic elements at nanometer scale. EMCD is a counterpart of X-ray magnetic circular dichroism (XMCD), a difference spectrum of the two X-ray absorption fine structure (XAFS) spectra obtained by clockwise and counterclockwise circularly polarized photons. More than 10 year have passed since Prof. Schattschneider of Vienna Technical University first proposed its idea, and the EMCD measurement scheme has developed as far as the stage where even atomic-plane resolution is feasible after a pile of trials and errors both from theoretical and experimental points of view. In this review we start with the fundamental principle of the measurement scheme and historical development, followed by several application examples mainly achieved by the present authors’ group. Finally we briefly touch the present status and prospects of the field in future.
Electron microscopy, once essential in diagnostic dermatopathology and investigative dermatology for skin diseases, has been overtaken by immunohistochemistry in the field of dermatopathology. However electron microscopy is important in the dermatology field even today. Here I present some excellent examples providing the importance of electron microscopy, viz., demonstrating the ultrastructure of intracytoplasmic canaliculi in porocarcinoma, fibronexus junction in atypical fibroxanthoma, lipid droplets in sebaceous carcinoma, tonofilaments in pseudovascular squamous cell carcinoma, inclusion bodies in Fabry disease, keratinization disorders and inherited hair diseases, among others, thereby indicating its utikity even today. Additionally, I would like to describe how electron microscopy can play an important role in the investigation for histogenesis of new diseases and the risk assessment for new industrial products.
Immunogold labeling for electron microscopy is a powerful technique to identify the localization of molecules in tissues and cells. Sample preparation of plants requires careful methods because plant cells have specific organelles that are difficult to fix and to infiltrate resin into, such as thick cell walls, large vacuoles and hard starch granules. In this paper, we introduce the techniques and methods for immunogold labeling of plant organs, tissues and cells. Moreover, we give some examples of immunogold labeling using plant samples.
The general treatment for a dental caries is to remove the decay and put in a filling artificial materials. Recently, tooth color composite resin is often used for filling material due to meet patients’ esthetic demand. Composite resin doesn’t adhere tooth itself, so dental adhesive is needed. It is known that the components of dental adhesive affect the longevity and clinical performance. We focused the interface between tooth and adhesive using transmission electron microscopy. Furthermore, we investigated the interaction functional monomer in dental adhesive and hydroxyapatite using X-ray diffraction (XRD), solid state nuclear magnetic resonance (NMR).
An advantage of fast in situ observation by a direct electron detection camera in materials science researches was confirmed by a research on crystallization process in amorphous antimony nanoparticles. At the early stage of the crystallization in amorphous nanoparticles, small crystalline nucleus on the surface repeats between formation and annihilation. When the nucleus size becomes more than the critical size, the crystal growth takes place in the whole nanoparticle. The growth rate depends on the particle size, and it was shown that the smaller the particle size is, the faster the growth rate is. It was suggested that the crystallization driven by long range elastic interaction energy due to small crystalline nucleus formation in amorphous nanoparticles is induced by short range atomic rearrangements.