Our Grant-in-Aid for Transformative Research Areas: “Cross-Scale Biology,” started in 2021. In this area, our researches focus on the 20–500 nm scale in cells, which has been difficult to approach in the past. Cryo-EM is one of several methods to observe phenomena and structures at this scale. In this article, in the context of “Cross-Scale Biology,” I will mainly discuss the analysis of intracellular complexes by cryo-EM/single particle analysis and cryo-electron tomography (hereinafter referred to as Cryo-EM/Tomo).

Atomic force microscopy (AFM) is an important analytical tool in biology because it allows direct observation of the movement of biomolecules in liquids. However, most conventional molecular-scale observation techniques using AFM are effective only for model systems constructed outside the cell on a substrate and cannot directly observe phenomena inside living cells. We developed nanoendoscopy AFM to solve this problem. In this technique, a long, thin needle probe is inserted into a living cell to directly observe intracellular structures and phenomena with AFM. So far, the internal structure of the entire cell, 3D distribution of actin fibers, and 2D nanodynamics of the inner surface of a cell membrane have been observed. Unlike previously proposed intracellular AFM measurement techniques, this technology can directly interact the probe with the intracellular structures. Therefore, major AFM measurements such as molecular resolution observation, mechanical property measurement, and molecular recognition observation can be realized in principle, and it is expected that this technology will be used for research on various intracellular phenomena in the future.

In the Transformative Research Area (A) “Cross-Scale New Biology,” we utilize quantitative cross-scale measurements from the molecular to the organelle and cellular levels, especially the measurement of meso-entangled bodies of 20–500 nm in size within cells, to elucidate the molecular and cellular mechanisms of life phenomena and diseases. In this section, we will introduce examples of how these cross-scale measurements have been used to gain a deeper understanding of fundamental life phenomena regarding intracellular protein molecular structure, localization, and dynamics and how the findings have been applied to elucidate disease mechanisms from a medical perspective.

Owing to the recent progress in microscopic technologies such as super-resolution microscopy and cryo-electron microscopy and the development of new methodologies for preparing observation samples without damages and labeling target molecules with high specificity, increasing numbers of attempts have been made to observe the fine structures of intracellular bodies and the dynamic behaviors of intracellular molecules. This review article introduces the latest topics of studies revealed by such cutting-edge imaging technologies, namely, (i) fine structure of the endoplasmic reticulum (ER) and distribution of a Ca²⁺ pump SERCA2b on the ER membrane, (ii) transport and incorporation of ferritin liquid droplets to the lysosome via macroautophagy and microautophagy, (iii) molecular-level observation of cellular fine structures in tissues, and (iv) formation and disaggregation of amyloid fibrils of a yeast prion protein Sup35. These observation targets are “meso-scale complexes” with diameters of 20 to 500 nm, and new insights into their intercellular structures and dynamics are being gained.

Visualization of the dynamics on the living cell surface is essential for understanding cellular functions such as migration, cancer invasion, and differentiation. In order to evaluate cellular dynamics, it is necessary to develop visualization techniques with nanoscale spatial resolution and non-invasion. Scanning ion conductance microscopy (SICM) is one of the most powerful microscopy techniques for obtaining fragile cell morphology under physiological conditions. In recent years, several research groups have improved the temporal resolution of SICM to visualize rapid cellular reactions. Here, we provide an overview of SICM, including the basic scanning mode and equipment, and recent researches on the development of high-speed SICM.

In the pursuit of exploratory nano- or atomic-scale devices, there is great demand for characterization of extremely small one- and two-dimensional structures. Imaging, fabricating, and measuring of local electronic properties of these structures can be performed with a one-probe scanning tunneling microscope (1P-STM). Yet, assessing the electrical conduction properties lateral to the surface demands two-probe (2P-) and four-probe (4P-) STM. The conductance measurement with 4P-STM is generally preferred over 2P-STM as it can eliminate the uncertainty in the probe-to-surface contact resistance that are derived from Schottky barrier. However, if we can solve the issue of the high resistive contact resistance with 2P configuration, we would be able to measure surface state conduction with the minimum number of probes, reducing arduous tasks in this field. In this report, we report the refinement of 2P-STM for surface state conduction measurements by Ohmic contact between probe and surface. We also utilized STM lithography to create electronically isolated regions from the otherwise surface area, finding that we can measure their conduction properties correctly by the Ohmic 2P-STM. Since the probe-to-probe distance can be reduced down to 30 nm, the present method will be useful in a wide range of fields of material sciences.

Secretory and membrane proteins synthesized at the endoplasmic reticulum are transported to the Golgi apparatus where they undergo post-translational modifications before being sorted and delivered to their final destinations: the plasma membrane, endosomes, or lysosomes/vacuoles. Recent advances in fluorescence microscopy have allowed us to visualize the detailed dynamics of membrane traffic in living cells, and a number of new concepts have been proposed. In this paper, we review recent findings on membrane traffic mechanisms revealed by super-resolution confocal live imaging microscopy (SCLIM), which we developed.