Volume EM Techniques

Focused Ion Beam Scanning Electron Microscopy

High-Resolution, Isotropic Volume Data for Accurate 3D Reconstructions

Highest Z-resolution
Isotropic 3D measurements
Reconstruction of subcellular features in accurate 3D proportions

Volume EM with Focused Ion Beam SEM (FIB-SEM)

With FIB-SEM, the resin-embedded sample is imaged with the SEM and then a focused ion beam mills away as little as 3-10 nm before it is sequentially imaged again. The very small step size in Z can be calibrated to the XY resolution of the SEM for isotropic 3D imaging, making FIB-SEM an excellent choice for accurate, ultrastructural 3D measurements for applications such as subcellular features or neuron connections.

Schematic Representation of a Typical Workflow

1

A trench is milled into a resin-embedded sample with a focused ion beam until the structure of interest becomes visible.

2

The newly exposed sample surface of the structure of interest is imaged. This milling and imaging process is repeated until the structure is completely imaged.

3

The acquired EM images are processed and digitally aligned into a 3D data set. Cell compartments can be identified and segmented.

4

The segmented 3D data set can be visualized, investigated, and statistically analyzed.

New Discoveries from the Ultrastructure of Life Virtual Seminar Series | January – June 2024

In a series of six webinars, explore the technological underpinnings of Volume EM imaging and its growing number of application areas in neurobiology, cancer research, developmental biology, plant science, and more.

Learn about vEM-specific sample preparation and technologies (array tomography, serial block-face SEM, and FIB-SEM), advanced image processing, data analysis, and result visualization capabilities of workflow-oriented software solutions.

Application Examples

High-Resolution, Isotropic Visualization of Cellular Ultrastructure in 3D

Data set kindly provided by Anna Steyer and Yannick Schwab, EMBL Heidelberg, Germany

3D Imaging of HeLa Cells

Automated 3D Serial Imaging with ZEISS FIB-SEM Technology

The focused ion beam was utilized to sequentially remove 8 nm thick layers of the specimen while the exposed block-face is scanned with a scanning electron microscope, thus obtaining a high-resolution 3D volume image. Automated segmentation and visualization of cellular components was done using an arivis Cloud-trained deep learning model in arivis Pro so that the different cellular components could be visualized and quantified.

3D reconstruction of algal Golgi body based on FIB-milling raw data — Image courtesy of Dr Louise Hughes, Oxford Brookes University, UK

Characterizing the Golgi Apparatus

To Better Understand Its Role in Protein Modification and Transport

This image shows a 3D reconstruction of algal Golgi body from a FIB-SEM data set. The data set distinguishes between the cis and trans faces of the Golgi (yellow/red: cis-golgi, purple/blue: trans-golgi). Segmentation of the cellular components from the high-resolution data sets acquired using ZEISS Crossbeam FIB-SEM technology ensures that internal components can be accurately characterized and quantified.

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