Volume EM with Focused Ion Beam Scanning Electron Microscopy
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

FIB-SEM milling

1

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

FIN-SEM Image acquisition

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. ​

Processing segmentation

3

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

3D visualization analysis

4

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

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 APEER-trained deep learning model in  Vision4D 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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