Electron tomorgraphy (ET) is a technique that retrieves three-dimensional (3D) structural information from a tilt series of two-dimensional (2D) projections that is imaged at several different angles. This method provides nanometer resolution from thick samples (100 nm to 1 mm) to provide more information about the depth of the sample.
While the electron beam is stationary, the specimen holder can rotate and the specimen can be tilted at 1 – 2° angular increments over a range of ± 70 degree about a fixed axis. To prevent radiation damage to the sample, the cumulative electron dose must be kept within tolerable limits.
Equipment and Software:
FEI Tecnai G2 Twin TEM – to collect a tilt series of 2D projections
Inspect 3D software – to reconstruct 3D volume
Amira software – to align the tilt series and perform modeling
Sample Preparation:
Regular TEM sample preparation
Section thickness is in the range of 100 nm – 1 mm and thicker than normal sections
Challenges:
Optimizing Data Collection
– Staining conditions
– Pre-irradiation needed (sections may shrink due to the beam intensity)
Image Data Set Alignment
– Calculation of alignment shifts
– Adjustment of tilt axis
Publications:
Asghari, P., Scriven, D. R., Ng, M., Panwar, P., Chou, K. C., van Petegem, F. and Moore, E. D. (2020). Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification. eLife, 9, e51602.
Baldelli, A., Trivanovic, U., and Rogak, SK. (2019) Electron tomography of soot for validation of 2-D image processing and observation of new structural features. Aerosol Science and Technology, 1-8.
Lyon, K., Adams, A., Piva, M., Asghari, P., Moore, ED., and Vogl, AW. (2017) Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells. Biology of Reproduction 96 (6): 1288-1302.