FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Abstract

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis

“…For FIB/SEM, the signal of the in-lens EsB detector is standard. Approaching the resolution limit, applying the inlens SE signal has several advantages: better resolution, better signal to noise ratio and much shorter exposure times (Villinger et al 2012). With an increased heavy metal impregnation (rOTO), high-resolution images (3072 × 2048 pixel) can be taken within 17–23 s, compared to the EsB signal with 30–50 s (Fig.…”

Structural and molecular bases of rod photoreceptor morphogenesis and disease

“…An initial demonstration of the application of focused-ion-beam milling with more conventional SEM of heavily fixed retina showed great promise for elucidating rod cell structure (Mustafi et al, 2011), but suffered from the defects, e.g., in disk spacing, observed in other samples fixed and stained for conventional TEM. These results suggest that combining ion beam milling of samples subjected to high pressure freezing with cryo-ET (Villa et al, 2013) or with serial scanning electron microscopy using detection of secondary electrons (Villinger et al, 2012) will add substantially to our understanding of rod structure in normal and diseased retinas.…”

Morphomics: An integral part of systems biology of the human placenta

“…The approach permits analysis of relatively large volumes such as whole cells. When combined with cryofixation (high-pressure freezing) and freeze substitution, FIB/SEM allows near-TEM resolution [93].…”