followed as in the instrument operation procedure (SOP) while
tilting the stage, such as holder type selection and tilting
mechanical limit.
10. Electron-beam radiation damage is an inevitable activity in a
TEM, especially to soft materials. It can introduce structural
change (loss of crystallinity or radiolysis of matter, etc.) and
mass loss [10]. Bubbling and charging induced by beam irradiation are the phenomena most frequently observed for
biological materials embedded in water or vitrified ice
[11]. Low dose imaging, minimizing the beam dose (beam
current density times exposure time), is an approach that can
be used to mitigate the radiation damage in a broad beam TEM
imaging for frozen specimens.
11. The quality of cryo-TEM specimen, such as the ice crystallinity,
thickness of vitreous ice, sample viscosity and concentration,
uniformity spanning the holes and grids, and the immunogold
labeling effectiveness, affects TEM imaging and analysis. The
surface property of TEM grids, the hole size, the material of
TEM grids, the cryofixation approach and conditions (plunge
speed, temperature, blotting parameters and cryogen, etc.) and
the procedure of cryo-EM specimen transferring are the main
influencing factors.
12. This step is to optimize the imaging conditions which comprise
spatial resolution, particles in the field of view and radiation
damage to obtain high quality TEM micrographs for frozen
specimens.
13. The Gatan cryo-transfer holder 914 allows the maximum tilt to
Æ90
at X-axis direction in our TEM column with a large gap
polepiece. However, the shadow of holder tip and TEM grid
bars limit the effective tilting angles to Æ75
. The position of
the area of interest may also limit the effective tilting angle but
nevertheless the maximum tilting should be tried to minimize
the missing wedge.
14. Other reconstruction software, such as IMOD, an open source
software, can also open the tilt-series of 2D micrograph, reconstruct and display the reconstructed 3D results.
15. Fiducial markers, such as colloid gold nanoparticles, are often
applied while preparing cryo-EM specimen for tracking and
alignment in tomography [12]. In our case, no extra fiducial
markers were introduced since the free antibody conjugated
with 1.4 nm nanogold was not removed during cryo-EM
specimen preparation.
510
Hui Qian et al.
tilting the stage, such as holder type selection and tilting
mechanical limit.
10. Electron-beam radiation damage is an inevitable activity in a
TEM, especially to soft materials. It can introduce structural
change (loss of crystallinity or radiolysis of matter, etc.) and
mass loss [10]. Bubbling and charging induced by beam irradiation are the phenomena most frequently observed for
biological materials embedded in water or vitrified ice
[11]. Low dose imaging, minimizing the beam dose (beam
current density times exposure time), is an approach that can
be used to mitigate the radiation damage in a broad beam TEM
imaging for frozen specimens.
11. The quality of cryo-TEM specimen, such as the ice crystallinity,
thickness of vitreous ice, sample viscosity and concentration,
uniformity spanning the holes and grids, and the immunogold
labeling effectiveness, affects TEM imaging and analysis. The
surface property of TEM grids, the hole size, the material of
TEM grids, the cryofixation approach and conditions (plunge
speed, temperature, blotting parameters and cryogen, etc.) and
the procedure of cryo-EM specimen transferring are the main
influencing factors.
12. This step is to optimize the imaging conditions which comprise
spatial resolution, particles in the field of view and radiation
damage to obtain high quality TEM micrographs for frozen
specimens.
13. The Gatan cryo-transfer holder 914 allows the maximum tilt to
Æ90
at X-axis direction in our TEM column with a large gap
polepiece. However, the shadow of holder tip and TEM grid
bars limit the effective tilting angles to Æ75
. The position of
the area of interest may also limit the effective tilting angle but
nevertheless the maximum tilting should be tried to minimize
the missing wedge.
14. Other reconstruction software, such as IMOD, an open source
software, can also open the tilt-series of 2D micrograph, reconstruct and display the reconstructed 3D results.
15. Fiducial markers, such as colloid gold nanoparticles, are often
applied while preparing cryo-EM specimen for tracking and
alignment in tomography [12]. In our case, no extra fiducial
markers were introduced since the free antibody conjugated
with 1.4 nm nanogold was not removed during cryo-EM
specimen preparation.
510
Hui Qian et al.