Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Principle of ATUM-FIB as a Hybrid Volume EM Approach Schematic of existing techniques for targeted volume SEM and comparison with the new ATUM-FIB approach (middle). Both ATUM and ATUM-FIB are microtomy-based, but ATUM-FIB generates semithick sections that can be selected by serial section light microscopy and SEM and subjected to further FIB-SEM investigation.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Comparison, Light Microscopy

Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Targeting Regions of Interest by Semithick Sectioning (A) Cartoon of the information content of BSD surface imaging and the potential FIB-SEM analysis of sections of different thickness. (B and C) (B) Transmitted light and (C) BSD images of semithin 0.5-μm and semithick 2-, 5-, and 10-μm sections of mouse brain tissue. cc, corpus callosum; ct, cortex; CA1, hippocampus CA1. Arrowheads indicate cracks, mainly in heavily stained tissue (corpus callosum). Scale bars 100 μm (top), 10 μm (bottom). (D) Consecutive (upper, lower row) semithick (2, 5, 10 μm) sections allow tracing cell bodies and blood vessels across sections. Somata and blood vessels that can be followed from one to the next section are colored blue; others are shown in orange. Black scale bar 10 μm; white scale bars 2 (left), 5 (middle), and 10 μm (right). See Figure S1 for similarity of opposing surfaces of consecutive sections.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Imaging, Staining

Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Generation of Semithick Sections by Precuring and Elevated-Temperature Microtomy (A) Comparison of resin performance for semithick sections of mouse cortex tissue. BSD images were acquired of 2 (top) and 5 μm (bottom) semithick sections of epon and LX112 blocks cured for 48 or 10 h and sectioned at room temperature or at 40°C. Left to right: epon cured for 48 h, sectioned at 24°C; LX112 cured for 48 h, sectioned at 24°C; LX112 cured for 10 h, sectioned at 24°C; LX112 cured for 48 h, sectioned at 40°C. Scale bar, 5 μm. Arrowheads indicate major (filled) and minor (hollow) tissue cracks. (B) Photographs of infrared lights installed at the sides of the microtome sample arm to warm the sample during semithick sectioning (left). The heated diamond knife is depicted with a lower heater and the upper sensor sticks and the temperature control (right). he, heating element; ir, infrared light; sa, sample arm; ta, tape; tac, tape collector; tec, temperature control; ts, temperature sensor. See Figure S2 for details on the heated diamond knife construction.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Comparison, Control

Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Serial Semithick Sectioning for Targeted FIB-SEM. Serial 5-μm-thick sections of mouse cortex were collected on CNT tape. (A) Overview of several consecutive sections on tape (left) and a single transmitted light micrograph (20x objective, right). Scale bars, 2 mm and 100 μm, respectively. (B) Transmitted light (left) and BSD images (10 × 10 × 5,000 nm resolution) of a selected region on a single section shown in (A) and maximum projection of all 93 sections revealing the vasculature morphology (right). Scale bar, 20 μm. (C) A random cortical FIB target site was chosen (blue box) (left). The selected region was prepared for FIB-SEM by carbon deposition and trench milling (right). Scale bars, 100 and 10 μm, respectively. (D) Cross section after FIB-SEM preparation showing the section—CNT tape adhesion site and the surface covered by a carbon layer (top). Defects in the surface layer topology are highlighted (black arrowheads). After the FIB-SEM run an xy section was reconstructed (bottom) from 2000 SE images (resolution 5 × 5 × 5 nm 3 , bottom). Scale bar, 1 μm. (E) 3D model of the FIB-SEM stack revealing myelinated axons (yellow), post synaptic densities (blue), and neuronal nuclei (gray). Scale bar, 2 μm. (F) Schematic of the ATUM-FIB strategy: sequential resin (LX112) curing and heated ultramicrotomy are required for semithick sectioning. Serial sections are attached onto glass slides for light microscopy and remounted onto silicon wafers for serial SEM imaging and target selection. A section of interest is remounted, adhered onto a standard sample holder, and carbon-coated for FIB-SEM examination. See Figure S2 for photographs of the different processing steps.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Light Microscopy, Imaging, Selection

Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Targeted FIB-SEM on Selected Consecutive Sites of Semithick Sections in Familial Alzhiemer's Disease (FAD) Cortex Samples (A) Schematic of ATUM-FIB on consecutive 5-μm-thick sections of mouse FAD cortex samples. Microglia (blue) contacts to plaques (orange) are targeted. (B) Transmitted light image of one section (20x objective, left) and average projection SEM of 18 sections (middle) revealing plaques (arrowheads). The 3D model (right) was segmented from a set of 36 serial transmitted light images. Blood vessels (gray), plaques (yellow), and the target plaque (orange) are highlighted. Scale bar 100 μm. (C–E) Schematics showing the imaging planes (gray) in the respective subfigures. (C) Three consecutive 5-μm-semithick sections of the FAD cortex sample on CNT tape. The microglial cell (blue), dystrophic neurites (orange), and amyloid core (yellow) are highlighted. The white box indicates the magnified image in (D). Scale bar, 5 μm. (D) Magnified surface image of the microglia to plaque contact site of section 1 (left). This region was targeted for FIB-SEM at 5 × 5 × 5 nm resolution. Reconstructions of two deeper levels within the semithick sections are shown (middle, right). Scale bar, 1 μm. (E) Stitched cross sections of corresponding areas in consecutive semithick sections 1 and 2 after FIB-SEM. Scale bar, 500 nm. (F) Region of interest of the selected section 1 of a FIB-SEM run. The 3D model shows the ER network (yellow) opposing the microglial plasma membrane (blue). Lysosomal structures are shown in light gray and mitochondria in dark gray (top). Scale bar, 500 nm. Representative SEM image revealing ER structures (black arrowheads) at sites of contact between microglia and dystrophic neurites (bottom). Scale bar, 200 nm.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Imaging, Clinical Proteomics, Membrane

Journal: iScience

Article Title: Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution

doi: 10.1016/j.isci.2020.101290

Figure Lengend Snippet: Comparison of Volume SEM Techniques for Targeting Rare Events Structures of interest like cell-cell interactions (microglia, magenta, dystrophic neurite, ocher) can be visualized by volume EM techniques. Current approaches for targeted imaging involve ATUM and FIB-SEM. ATUM-FIB combines the advantages of both approaches. High-precision targeting based on ultrastructural features is enabled, whereas increased section thickness reduces screening time and complexity compared with ATUM. Targeted FIB-SEM targeting is usually based on microCT data with limited xy resolution combined with trimming (arrows). A 500 μm z depth would be covered by 100 semithick sections (at 5 μm thickness, ATUM-FIB) instead of 10,000 ultrathin sections (at 50 nm thickness, ATUM). Comparable with FIB-SEM, ATUM-FIB allows for acquisition of the region of interest (black box) at isotropic voxels.

Article Snippet: Here, we developed a multiscale method for targeted FIB-SEM on semithick (2–10 μm) sections named ATUM-FIB.

Techniques: Comparison, Imaging