Holotomographic microscopy (HTM) measures the refractive index (RI) tomograms of living cells and tissues in three dimensions. The ability to observe biological processes at high spatial and temporal resolution opens uncharted territories for cell biologists, however, current HTM devices have a limited throughput. We show here the first automated multi-well plate-compatible HTM device, the CX-A. Thanks to state-of-the-art environment control and a new type of autofocus, the CX-A can record multiple conditions in parallel over large fields of view, while its software EVE supports automated single-cell segmentation and quantification. This opens the door to new applications for HTM, from drug screening to systems biology.

In correlative light and electron microscopy (CLEM) workflows, identifying the same sub-cellular features in tissue by both light (LM) and electron microscopy (EM) remains a challenge. Furthermore, use of cryo-fixation for EM is desirable to capture rapid biological phenomena. Here, we describe a workflow that incorporates cryo-confocal laser scanning microscopy into the CLEM process, mapping cells in brain slices to re-image them with serial section scanning electron microscopy (ssSEM) array tomography. The addition of Airyscan detection increased the signal-to-noise ratio (SNR), allowing individual spines in thick frozen tissue to be visualized at a sufficient spatial resolution, providing a new tool for a CLEM approach to capture biological dynamics.

Electron microscopy has enabled atomic resolution imaging of matter. However, unlike optical spectroscopic imaging, traditional electron microscopes provide limited spectroscopic information in terms of their energy resolution. Only recently, owing to advances in monochromated STEM-EELS, have transmission electron microscopes (TEMs) been able to attain a high energy resolution. We recently proposed combining spectrally selective photoexcitation with HRTEM to achieve sub-nanometer scale optical imaging, a technique we called photoabsorption microscopy using electron analysis (PAMELA). To realize PAMELA-TEM experimentally, we constructed a TEM holder with an optical feedthrough, capable of photoexciting materials with different wavelengths. In this article, we describe our process for designing and fabricating an optical TEM specimen holder, highlighting important aspects of the design.

With the growing applications of temporally resolved electron microscopy for probing basic phenomena and reducing beam-induced damage, a multifaceted introduction to the field of ultrafast transmission electron microscopy is provided. This primer includes techniques and equipment as well as implementation perspectives. Historical developments and recent technical advances will provide insight into ultrafast capabilities for research as well as educate electron microscopists on the general techniques. This technology review also includes applications enabled by ultrafast techniques using various sample stimuli from multidisciplinary fields.