7. In SerialEM, the “Low Dose” function is used such that search mode is done at
very low “magnification” (i.e., extreme undersampling over a large field of
view). Focussing is done at a distance away from the site of interest, along the
tilt axis, at very high magnification over a small field of view so that fiducial
gold particles are easily brought into focus by the autofocus module in
SerialEM.
8. Exposure conditions are typically 2048X2048 arrays, sampled at 2–5 ls per
dwell spot. This means that for each tilt, the acquisition time is about 8–20 s per
frame.
9. Datasets are reconstructed using a variety of software, including IMOD [108],
Tomo3D [109] and TomoJ [110].
2.5.2 Plastic section data collection
1. Sections are mounted on Formvar coated 50–200 mesh grids. For STEM
tomography, slot grids are avoided; while grids mounted on slot grids may be
stable during TEM tilt series acquisition, they tend to move during STEM
acquisition and the resulting images may be distorted.
2. Grids are coated with carbon on both sides, and then floated on poly lysine
solution (1 mg/ml), washed with bidistilled water, floated on colloidal gold
solution and washed again. The procedure is repeated for the other side of the
grid. Incubation time and concentration of solutions can be adjusted for optimal
distribution of colloidal gold. Grids can be plasma cleaned or glow discharged
prior to incubation with poly lysine if the distribution of gold particles is too
sparse.
3. Grids are double stained with 2% uranyl acetate in 50% ethanol and Reynolds
lead citrate for 10 min each step. Ethanolic uranyl acetate solution is preferred in
order to enhance the infiltration of uranyl into the thick section. Note that while
stain provides contrast it also adds to the beam broadening effect. Hence, heavy
stain of thick sections may in principle be counterproductive and the staining
procedure may need to be optimized accordingly.
4. Gently plasma-clean the grid in the side-entry holder before insertion. This is
mandatory for STEM acquisition for two reasons: a. Dirt on the holder may
deflect the beam due to charging effects. b. Contamination on the surface of the
sample builds-up during scanning, which results in darkening and blurring of
the image.
5. After inserting into the microscope, irradiate the sample with a bright wide-field
electron beam at very low magnification for 30 min.
6. In TEM imaging mode, appropriate areas for study are selected, and those
positions are saved.
54
S. G. Wolf et al.
very low “magnification” (i.e., extreme undersampling over a large field of
view). Focussing is done at a distance away from the site of interest, along the
tilt axis, at very high magnification over a small field of view so that fiducial
gold particles are easily brought into focus by the autofocus module in
SerialEM.
8. Exposure conditions are typically 2048X2048 arrays, sampled at 2–5 ls per
dwell spot. This means that for each tilt, the acquisition time is about 8–20 s per
frame.
9. Datasets are reconstructed using a variety of software, including IMOD [108],
Tomo3D [109] and TomoJ [110].
2.5.2 Plastic section data collection
1. Sections are mounted on Formvar coated 50–200 mesh grids. For STEM
tomography, slot grids are avoided; while grids mounted on slot grids may be
stable during TEM tilt series acquisition, they tend to move during STEM
acquisition and the resulting images may be distorted.
2. Grids are coated with carbon on both sides, and then floated on poly lysine
solution (1 mg/ml), washed with bidistilled water, floated on colloidal gold
solution and washed again. The procedure is repeated for the other side of the
grid. Incubation time and concentration of solutions can be adjusted for optimal
distribution of colloidal gold. Grids can be plasma cleaned or glow discharged
prior to incubation with poly lysine if the distribution of gold particles is too
sparse.
3. Grids are double stained with 2% uranyl acetate in 50% ethanol and Reynolds
lead citrate for 10 min each step. Ethanolic uranyl acetate solution is preferred in
order to enhance the infiltration of uranyl into the thick section. Note that while
stain provides contrast it also adds to the beam broadening effect. Hence, heavy
stain of thick sections may in principle be counterproductive and the staining
procedure may need to be optimized accordingly.
4. Gently plasma-clean the grid in the side-entry holder before insertion. This is
mandatory for STEM acquisition for two reasons: a. Dirt on the holder may
deflect the beam due to charging effects. b. Contamination on the surface of the
sample builds-up during scanning, which results in darkening and blurring of
the image.
5. After inserting into the microscope, irradiate the sample with a bright wide-field
electron beam at very low magnification for 30 min.
6. In TEM imaging mode, appropriate areas for study are selected, and those
positions are saved.
54
S. G. Wolf et al.