Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

Two-photon microscopy (2PM) provides three-dimensional (3D) and four-dimensional (4D) (x, y, z, t) imaging in living specimens or under experimental physiological conditions very close to live. In conjunction with fluorescent labels, 2PM provides a powerful means of investigating the relationships between structure and function at the microscopic level that are key to understanding biological systems. This technique is able to provide time-resolved, 3D images of dynamic systems with near-diffraction-limited resolution and highly specific structural contrast.

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 ~200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

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