For volume imaging of biological samples and preparation of lamella for TEM

In FIB/SEM microscopy, SEM is combined with Focused Ion Beam (FIB) milling, where an ion beam is focused into a small probe to remove material from the specimen's surface, after which the SEM images the exposed surface. The process is iterative, and as more layers are milled, a series of 2D images is acquired. These images can then be stacked together to generate a high-resolution 3D representation.

FIB/SEM is part of a range of techniques called Volume electron microscopy, enabling the visualization of the 3D ultrastructure of cells and tissues, routinely probing volumes with dimensions up to several tens of micrometers. By providing a comprehensive and detailed understanding of the spatial relationships and intricate three-dimensional structures in life science samples, volume imaging significantly enhances the capabilities of biological research. At Radboudumc EMC, we specialize in volume imaging using FIB/SEM technology and offer a range of imaging strategies for exploration of a wide variety of samples.

Cryogenic FIB/SEM

We specialize in performing 3D reconstruction of samples in a near-native, hydrated state using cryoFIB/SEM technology operating at -160 °C. Our approach begins by fixing the sample into a vitrified state through high-pressure freezing, followed by precise milling and imaging.

Targeted cryogenic FIB/SEM

A difficulty when analysing vitrified samples by FIB/SEM is the identification of the region of interest to be imaged within the whole sample. To guide us, we combine cryoFIB/SEM with cryo-fluorescence imaging to precisely identify the fluorescence labelled region of interest where 3D FIB/SEM should be carried out. 

We employ this technique to visualize the uptake of extracellular material into cytoplasmic vesicles, a process that requires observation in a hydrated environment. By maintaining the sample in a near-native state, we ensure the preservation of its inherent characteristics and enable detailed analysis at a subcellular level. 

Room Temperature FIB-SEM

At Radboudumc EMC, we are perform 3D reconstruction of resin-embedded biological samples using FIB/SEM technology. We are committed to designing the best sample preparation approach tailored to your specific sample, ensuring minimal alterations that may be caused by processing and staining procedures. Our facility offers a wide range of sample preparation techniques, including Vibratome sectioning, conventional resin embedding, high-pressure freezing, and automated freeze-substitution, to optimize the output and quality of your sample.

We recently used this approach to observe the 3D ultrastructure of Plasmodium falciparum gametocytes that were embedded in agarose, chemically stained and imaged with FIB-SEM. 

Cryo and room temperature lift-out

Lift-out is a specialized technique in FIB/SEM for extraction of a thin lamellar section from a specific region of interest (ROI) in a bulk sample for high-resolution transmission electron microscopy (TEM) imaging.

At Radboudumc EMC, we employ a Kleindiek microgripper for precise lamella preparation. This procedure can be applied to samples at room temperature, but also by cryo lift-out for vitrified frozen biological samples.

In a typical procedure, the ion beam in the FIB/SEM microscope is utilized to mill trenches around the targeted region, revealing the ROI. After milling, a micromanipulator equipped with a fine tungsten needle or specialized cryo-liftout needle is used to lift the targeted region from the rest of the sample. The extracted region is then transferred to an appropriate substrate or grid for subsequent (cryo)TEM analysis.

Our cryo lift-out workflow: localize ROI, mill three trenches with FIB, use microgripper to lift lamella.
Our cryo lift-out workflow: localize ROI, mill three trenches with FIB, use microgripper to lift lamella.