MST43

Scanning electron microscopy (SEM) is widely recognized as both a surface-imaging tool and an analytical platform, combining high-resolution observation with robust and scalable measurement capabilities. In biological research, however, SEM has traditionally been positioned as a tool for observing surface topography, while the investigation of internal ultrastructure has relied almost exclusively on transmission electron microscopy (TEM) using ultrathin sections as the standard approach. This long-standing division has recently begun to change. By fully exploiting the scalability, robustness, and accessibility of SEM, flat-surface histological imaging has enabled SEM to function as a platform for visualizing internal tissue architecture with TEM-like ultrastructural detail.

This methodological shift naturally led to the emergence of SEM-based volume electron microscopy (vEM). Techniques such as array tomography, serial section SEM, and FIB–SEM tomography allow systematic acquisition of serial images from consecutive sections, enabling three-dimensional reconstruction of tissue architecture over volumes far exceeding those accessible by TEM tomography. Using these approaches, we have visualized biological structures across a wide range of spatial scales, from the internal organization of organelles such as mitochondria to higher-order cellular and tissue networks, within tissue volumes on the order of approximately 10 to 200 µm in lateral dimensions. This intermediate spatial scale, which is difficult to access by conventional TEM tomography or three-dimensional light microscopic techniques, represents a critical regime for linking molecular organization to cellular interactions and tissue architecture(1, 2). We define this regime as the biological mesoscale.

An additional strength of flat-surface SEM imaging lies in its exceptional compatibility with correlative light and electron microscopy (CLEM). Optical imaging can be performed on the same sections prior to electron microscopy, allowing specific cells, structures, or molecular signals to be identified and subsequently examined at ultrastructural resolution. This seamless correlation between functional or molecular information and ultrastructure makes SEM a highly effective platform for integrative biological imaging, while remaining readily extendable to three-dimensional analysis through integration with SEM-based volume electron microscopy approaches (3, 4).

In this presentation, I will introduce the principles and workflows of flat-surface histological imaging, SEM-based volume electron microscopy (vEM), and CLEM, and highlight recent collaborative work using these methods (5). Finally, I will discuss how this redefined role of SEM complements cryo-EM by addressing the biological mesoscale, providing a forward-looking framework for understanding complex tissue organization across multiple spatial scales. 

References:

1.  Miyazono Y, Hirashima S, Ishihara N, Kusukawa J, Nakamura KI, Ohta K. Uncoupled mitochondria quickly shorten along their long axis to form indented spheroids, instead of rings, in a fission-independent manner. Sci Rep. 2018;8(1):350.

2.  Migita H, Rikimaru H, Rikimaru-Nishi Y, Koga N, Watanabe K, Ohta K, et al. Analysis of Scars and Keloids by Focused Ion Beam/Scanning Electron Microscopy: Distinguishing Between Hypertrophic Scars and Keloids. Ann Plast Surg. 2020;84(4):379-84.

3.  Ohta K, Hirashima S, Miyazono Y, Togo A, Nakamura KI. Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM. Microscopy (Oxf). 2021;70(2):161-70.

4. Kamegai R, Uenosono A, Ikeda F, Ohta K and Yanagitani K: Mammalian cells evacuate and shelter mitochondrial DNA from destruction following hypoxia response-induced mitophagy. bioRxiv:2025.08.01.667885,  Pre-print (2025)

5.  Hipkaeo W, Ohta K, Kondo H. Three?Dimensional Ultrastructural Characterization of Fibroblastic/Stromal Cell Processes in Mouse Ureteral Lamina Propria by FE?SEM Array Tomography. Microsc Res Tech. 2025.