Electron Tomography
Why ELectron Tomography is important?
Electorn tomography data collection (Plastic sections)
Electon tomography reconstruction and generating tomographic model
Structure analysis and quantitative data extraction from tomographic model
Image gallery for Electron Tomography research projects
The basic principle of the Electron Tomography has been developed since 1968 by DeRosier & Klug*. Electron Tomography is a method to reconstruct a 3-dimensional image of a specimen from 2-dimension electron micrographs. The fundamental idea is that the 3-dimensional transform of an object can be built up by using Fourier transforms of 2-dimensional projected views of the object obtained over a wide range of viewing directions. When there is sufficient data, the 3-dimensional image of the object can be reconstructed from the Fourier inversion of the resulting 3-dimensional transform. The most compelling point of Electron Tomography is the ability to obtain high-resolution (6-8nm) 3-dimensional structure information of the specimen, which is 20-30 times the resolution of serial thin section electron microscopy and 400-1000 times the resolution of confocal scanning laser microscopy. Electron Tomography has quickly become the most advance technology in cell biology to effectively define the 3-demensional architecture of cellular structures.
* DeRosier DJ, Klug A (1968) Reconstruction of Three Dimensional Structures from Electron Micrographs. Nature 217: 130-134.
Structure is always intimately related to function. In living organisms, structure not only reflects on function but also defines many functional attributes. The high resolution 3-dimensional structure information in molecular and even atomic level organization of cells and tissues provided by Electron Tomography is the basis of many functions. Electron Tomography is currently the best method for defining the 3-dimensional nanoscale architecture of cellular structures with complex morphologies. The nanoscale structural and quantities information derived from the Electron Tomography model enable us better understanding the complexity of organelle relationships and provide new information to previously established function postulates.
After fixation, the samples are embeded, polymerized and hardened in a plastic sample block and are ready for trimming and sectioning. Serial sections ( about 150 -200nm) are cut continously by a diamond knife by a ultra microtome and are collected on formvar-coated copper slot grids.
To obtain data for a dual-axis tomogram, the tilting serial images of each serial section are taken under Transmission Electron Microscope with a tilting angle range and decrement angles (eg. from-40o to +40o, 1o decrement). Then rotating the sample grid 90o in the sample rod, the tilting serial images for each serial section with same tilting angle range and decrement are collected. The tilting angle range and decrements setup, image focus, tilt, alignment and capture during the process are all controlled by the software.
For example:
Here is six serial sections of the Golgi apparatus in a Penium cell (a unicell Algae). Tilting angle ranges from 鈭�55掳 to 55掳 in 1掳 increment for each section. The following images shows the TEM images at 0 degree of each serial section.
For each serial section, there are two sets of image stack with images from different angles rotated around two orthogonal axes. The following steps are used to complete tomography reconstruction and generate model:
- Each section鈥檚 single axis tomograms are reconstructed by etomo software interface in the IMOD software package (Kremer et al.1996).
- Two orthogonal single axis tomograms were merged into one with a warping procedure (Mastronarde 1997). Step one & two process was repeated for all serial sections.
- All the section tomograms are joined by manually aligning larger structures from the bottom of one tomogram to the top of the adjacent tomogram using midas in the IMOD software. Then tomogram join was refined by fiducial markers on trajectories features though the adjacent tomograms (Ladinsky et al. 1999).
- After completing the tomogram reconstruction, the 3-dimensional model of joined tomogram was built in 3dmod software interface, which we refer as 鈥渕odeling鈥�. In modeling, each specific cellular structure is considered as an individual 鈥渙bject鈥�, which is assigned in a specific color. Different objects are assigned in different colors. In every tomogram slice, each object contour is carefully manually traced by a graphic pad. In order to make sure the accuracy of the modeling, the contour tracing of an object normally starts at a tomogram slice where the object was most distinct, then continue through adjacent slices in both direction.
- At last, after all the contours have been drawn on all the consecutive tomogram slices, imod mesh which uses a mesh triangular computation to define the surface of each object, was applied to achieve a smooth 3-dimensional surface for each object. The final complete model displays all the modled objects together.
Kremer JR, Mastronarde DN, Mcintosh JR (1996) Computer Visualization of Three-Demensional Image Data Using IMOD. Journal of Structural Biology 116: 71-76.
Mastronarde DN (1997) Dual-Axis Tomography: An Approach with Alignment Methods that Preserve Resolution. Journal of Structural Biology 120: 343-352
Ladinsky MS, Mastronarde DN, McIntosh JR, Howell KE (1999) Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144: 1鈥�16.
For example:
The below image on the left is one of the tomogram slices in one serial section after tomographic reconstruction. The image on the right is the tracing contoure for each cicsterna on the tomogram slice. Each cicsterna in the Golgi structure is a seperated object represented by a different color. (Scale bar is 200nm)
After all the contours were drawn on each tomogram slice of all the serial sections, The IMOD mesh tool was used to creat a 3D surface for each object. The entire model or individual structure in the model can be rotated and displayed in any direction. The below images are the whole model view of the Golgi apparatus in Penium in six directions. The overall view of tomographic model rotating along X axis and Y axis also presented in below video, which is the best way to display general 3D architecture. (Scale bar is 200nm)
Besides to reconstruct 3-dimension morphology of cellular structures, the other powerful point of electron tomography is to extract quantitative information from 3-dimension model. From imodinfo program, several structural parameter can be quantified and compared. For example, the below two tables show the surface area and volume comparison of twelve cisternaes in the above Golgi stack of Penium (cisternae 1 and cisternae 12 are on the cis side and trans side of the Golgi respectively). Quantitative data can significantly support structure result explaination and can improve the understanding of the connections between cellular structure and function.
The below videos are from recent Electron Tomography research projects on different green Algae samples. All the works are accomplished in 三亿体育官网 McGraw Microscopy Imaging Center.
"Fused" Golgi aparratus inside of Penium (a green Algae) cell. TEM image (left) and 3D electron tomography model (right).
Golgi aparratus inside of Chara (a green Algae) cell. TEM image (left) and 3D electron tomography model (right).
Endosidin 5 (Endosidins are a group of low-molecular-weight compounds used to target specific components of the endomembrane) treated Golgi aparratus inside of penium cell. The positioning and structure of the Golgi bodies in the cell are significantly changed comparing to the untreated cells. TEM image (left) and 3D electron tomography model (right).
BFA (a kind of endomembrane inhibitors) treated Golgi aparratus inside of penium cell. The structure of the Golgi bodies in the cell are altered comparing to the untreated cells. TEM image (left) and 3D electron tomography model (right).
Concanamycin A (a kind of endomembrane inhibitors) treated Golgi aparratus inside of penium cell. The architecture of the Golgi bodies in the cell are changed comparing to the untreated cells. TEM image (left) and 3D electron tomography model (right).
Endosidin 3 (Endosidins are a group of low-molecular-weight compounds used to target specific components of the endomembrane) treated Golgi aparratus inside of penium cell. Some of the Golgi bodies in the cell are changed comparing to the untreated cells. TEM image (left) and 3D electron tomography model (right).
