3D Electron Microscopy Imaging

The 3D electron microscopy imaging is available using Serial Block Face (SB-EM) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) or Electron Tomography (ET)

Electron tomography is TEM based method best suited for high resolution (~1-4 nm/pixel) imaging of relatively small areas. Typical dimensions of ET dataset is about 5.0 x 5.0 x 0.25 um.

Focused ion beam scanning electron microscopy offers isotropic high resolution imaging (from ~4 nm/pixel) of embedded specimen of moderate volumes.

Serial Block Face Scanning Electron Microscopy generates datasets with moderate resolution (~10-20 nm/pixel in the XY plane; 25-50 nm/pixel in the Z plane) covering large areas. Typical dimensions of SB-EM dataset is 40 x 40 x 40 um.
3D Serial Block Face Scanning Electron Microscopy Imaging

Location: Room 1415
installed on FEI Quanta 250 FEG 

Features:

  • Unique quality of the alignment allowing a perfect 3D visualisation
  • 3View allows automated acquisition of 3D ultrastructure by sequentially imaging a freshly cut, resin-embedded block face by a high-precision ultramicrotome within the SEM chamber.
  • Unlike the 3D imaging techniques in light microscopy, the spatial resolution provided by 3View in the Z direction is comparable to its X-Y resolution and does not degrade with depth.
  • The cut step size can be set from 20 to 200 nanometers.
  • The images are acquired from the block-face, not the sections which are discarded. Image capture is done using a backscatter electron detector.

Extras:

A custom script for batch conversion of DM3 and DM4 files to the TIF format. During conversion all metadata from the DM-files are extracted and stored in a text HDR (header) file.
The script is based on original BatchConvertToFixed script by John Minter, Eastman Kodak, Foundation Science and Technology Center 

 

How to preprocess images from Gatan 3View using DigitalMicrograph and MIB
Focused ion beam scanning electron microscopy

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is a 3D imaging technique that can be applied to biological tissues. It uses a scanning electron microscope equipped with a focused gallium ion beam to sequentially mill away the sample surface. In-column backscattered (EsB) and secondary electron (SE) detectors are used to image the milled surfaces. As a result, a large series of images can be automatically acquired and combined into a 3D dataset. FIB-SEM is suitable for imaging small to moderate volumes of biological samples with up to 4-nm isotropic voxels

 

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Electron Tomography

Electron tomography (ET) is a method for obtaining 3D-structural information using TEM. The images of a tilt series are acquired over a wide range of viewing angles, and then these 2D-projection images are aligned and back-projected to generate a 3D-reconstruction. In biological research, ET can be applied to study unique structures, which are in the context of a cell. ET is ideally suited to reveal cell organelles ultra structure in the 5-20 nm resolution range. 

The first movie (on the left) shows serial tilted views of the 120 nm thick section from a cell of Trypanosoma brucei . Images were collected (TEM Tecnai F20) at one degree intervals over a range from -62° to +62° at magnification of 11500x. Gold particles (10 nm) were used as fiducial markers to align the images of the raw dataset (H. Vihinen). 

The second movie (on the left) shows a serial section reconstruction of a fragment of a cell of Trypanosoma brucei (H. Vihinen). 

The third movie (on the left) shows the modeled Golgi complex and ER of a cell of Trypanosoma brucei. The membranes of rough endoplasmic reticulum are coloured as yellow and Golgi in shapes of blue (Eija Jokitalo and Ilya Belevich).

Image alignment and reconstruction are done utilizing IMOD software package (The Boulder Laboratory for 3-Dimensional Electron Microscopy of Cells, University of Colorado). The segmentation was done in Microscopy Image Browser, the final model visualized with Amira (ThermoFisher Scientific).