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
Regular TEM sample preparation
Section thickness is in the range of 100 nm – 1 mm and thicker than normal sections
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
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