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Volume EM with Cryogenic FIB-SEM​
Volume EM Techniques

Cryogenic FIB-SEM​

Capture a Snapshot of Cellular Dynamics at Near Native States in Ultraresolution​
  • Near native, artifact-free preservation of biological samples
  • Highest Z-resolution​
  • Isotropic 3D imaging

Volume EM with Cryogenic FIB-SEM

With FIB-SEM, the 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 3D measurements. Traditional chemical fixation of cells or tissues for electron microscopy can cause ultrastructural changes. Cryogenic fixation will preserve the sample at near native states and cryogenic FIB-SEM, which maintains the sample's low temperature, will capture a near native, 3D ultraresolution snapshot of cellular events. This can be combined with other cryo methods, such as light and confocal microscopy.​

Schematic Representation of a Typical Workflow​

Cryo FIB-SEM milling

1

A trench is milled into a vitrified, unstained sample with a focused ion beam until the structure of interest becomes visible.

Cryo FIB-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. The sample stays vitrified during the entire process.

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 Example

Understanding the Biomineralization Process of Calcite Crystals in a Coccolithophorid Alga

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S. Sviben & A. Scheffel, Max Planck Institute Plant Physiology, and L. Bertinetti, Max-Planck Institute of Colloids and Interfaces, Potsdam-Golm, Germany

Formation of Coccoliths in the Coccolithophore Emiliania Huxleyi​

Visualization of Calcite Particles and Their Ultrastructural Environment

The ultrastructural environment of soluble, amorphous calcium phases of coccolithophores is difficult to image with the classical, water-based preparation protocols. FIB-SEM operated under cryo conditions enables to image vitrified marine algae in the near-to-native state elucidating the ultrastructure in 3D.

E. huxleyi cells were high-pressure frozen. During the imaging process, the sample was kept in a frozen state; the FIB-SEM data set was acquired using a ZEISS Crossbeam under cryogenic conditions. The 3D reconstruction shows the mature coccoliths (yellow), a coccolith in statu nascendi (blue) and lipid bodies (red).

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