cryo- fluorescence microscopy [132–136], and time-resolved electron tomography of
cellular events [137].
1.14 Protocol
This protocol is universal in the sense that low-dose acquisition schemes should be
used for plastic sections as well as for beam-sensitive cryo samples. The reader
should consult several such protocols, including those published by the Boulder,
CO, group and the cryo protocol of Sun and Li [138].
(1) Prepare sample according to data acquisition strategy (plastic or cryo). For
detailed instructions on plunge freezing, refer to one of the numerous protocols,
e.g.: [139–141]. Carbon-coating of plastic sections is recommended [82].
(2) Perform all microscope column alignments, and in particular, beam tilt pivot
points, tomo rotation centre, beam tilt calibrations, image (beam) calibrations, image shift pivot points, and image shift for the working magnification
range.
(3) Record a gain reference and check that it is valid via integration and/or
auto-correlation (e.g. Digital Micrograph, Gatan, Pleasanton, CA). Determine
the electron dose.
(4) Load the sample. Plastic sections can be mounted in a cryo sample holder
where available. In this case, the holder is inserted, filled with liquid nitrogen, and allowed to reach a stable temperature before continuing.
(5) Locate the region of interest. This might be done manually or via input of
coordinates obtained via fluorescence microscopy on a Finder-style sample
grid, for example. Use the lowest practical magnification and enhance phase
contrast by underfocussing.
(6) Adjust the eucentric height, and focus on the specimen.
(7) Centre the condenser aperture and adjust the gain conditions.
(8) Align the rotation centre, preferably using gold markers.
(9) Correct for objective astigmatism, and focus on the specimen.
(10) Determine the possible tilt range, i.e. until a grid bar obscures the detector or
image quality is too degraded.
(11) Set the acquisition states for a minimum of tracking, focus and recording
positions. Set the focus and tracking positions adjacent to the recording position but further along the tilt axis where the illuminated areas do not overlap.
(12) Set the defocus value to correspond to the resolution of the target structure
(not applicable for phase plate -equipped instruments).
(13) If necessary, burn a hole in the tracking and/or focus areas by condensing the
beam for a few seconds. For stained plastic sections, specify focus close to
zero (say, −200 nm) to ensure that the autofocus function attempts to calculate a target focus value. For plastic sections, focussing and tracking can be
done on the target (recording) area but this is not recommended.
26
A. Leis
cellular events [137].
1.14 Protocol
This protocol is universal in the sense that low-dose acquisition schemes should be
used for plastic sections as well as for beam-sensitive cryo samples. The reader
should consult several such protocols, including those published by the Boulder,
CO, group and the cryo protocol of Sun and Li [138].
(1) Prepare sample according to data acquisition strategy (plastic or cryo). For
detailed instructions on plunge freezing, refer to one of the numerous protocols,
e.g.: [139–141]. Carbon-coating of plastic sections is recommended [82].
(2) Perform all microscope column alignments, and in particular, beam tilt pivot
points, tomo rotation centre, beam tilt calibrations, image (beam) calibrations, image shift pivot points, and image shift for the working magnification
range.
(3) Record a gain reference and check that it is valid via integration and/or
auto-correlation (e.g. Digital Micrograph, Gatan, Pleasanton, CA). Determine
the electron dose.
(4) Load the sample. Plastic sections can be mounted in a cryo sample holder
where available. In this case, the holder is inserted, filled with liquid nitrogen, and allowed to reach a stable temperature before continuing.
(5) Locate the region of interest. This might be done manually or via input of
coordinates obtained via fluorescence microscopy on a Finder-style sample
grid, for example. Use the lowest practical magnification and enhance phase
contrast by underfocussing.
(6) Adjust the eucentric height, and focus on the specimen.
(7) Centre the condenser aperture and adjust the gain conditions.
(8) Align the rotation centre, preferably using gold markers.
(9) Correct for objective astigmatism, and focus on the specimen.
(10) Determine the possible tilt range, i.e. until a grid bar obscures the detector or
image quality is too degraded.
(11) Set the acquisition states for a minimum of tracking, focus and recording
positions. Set the focus and tracking positions adjacent to the recording position but further along the tilt axis where the illuminated areas do not overlap.
(12) Set the defocus value to correspond to the resolution of the target structure
(not applicable for phase plate -equipped instruments).
(13) If necessary, burn a hole in the tracking and/or focus areas by condensing the
beam for a few seconds. For stained plastic sections, specify focus close to
zero (say, −200 nm) to ensure that the autofocus function attempts to calculate a target focus value. For plastic sections, focussing and tracking can be
done on the target (recording) area but this is not recommended.
26
A. Leis