(i) Stop the acquisition and save a series of images.
2. 3D Reconstruction and Visualization (see Note 14).
(a) Open a tilt series of 2D images recorded above in
Composer.
(b) Check the effective data to be used for further alignment.
(c) Select the interested region for prealignment.
(d) Pick fiducial markers within the selected region and perform fine alignment (see Note 15).
(e) Check the fine alignment results and perform the manual
alignment if necessary.
(f) Set reconstruction parameters and perform reconstruction. The filtered back projection (FBP) method was
used in our case.
(g) Export reconstructed result to Visualizer-kai. Perform
rendering to display the volume and sections of reconstructed 3D data (Fig. 7).
4 Notes
1. The lacey carbon film copper TEM grid is the most popular and
the cheapest grid to start with. The holes are variable which is
good for accommodating nonhomogeneous nanoparticles, but
the rigidity and stability of samples embedded in ice may not be
strong enough for some analysis due to the weak carbon support between holes. Extracontinuous carbon or formvar thin
film may therefore be needed for the enforcement of lacey
TEM grids.
2. The freshly prepared carbon films are hydrophilic, but they
become more hydrophobic over time. It is therefore necessary
to restore the surface hydrophilicity so that the aqueous solutions spread over the grid film easily and evenly. This can be
realized using plasma or glow discharging [9]. There are different types of glow dischargers on the market. But basically the
discharge current, pressure level, atmosphere gas and discharging time need to be tested to obtain the desired surface
property.
3. The successful application of immunogold labeling depends on
the capacity of antibodies to infiltrate throughout the lipid
membrane, and the specificity of recognition between
antigen-primary antibodies. The low antigen encapsulation in
archaeosomes also makes the labeling more challenging.
4. Labeling in a suspension method can be used. In this case,
primary Ab was added to an aliquot of archaeosomes in a vial
and incubated at 37
C for about 1 h. However, the subsequent
508
Hui Qian et al.
2. 3D Reconstruction and Visualization (see Note 14).
(a) Open a tilt series of 2D images recorded above in
Composer.
(b) Check the effective data to be used for further alignment.
(c) Select the interested region for prealignment.
(d) Pick fiducial markers within the selected region and perform fine alignment (see Note 15).
(e) Check the fine alignment results and perform the manual
alignment if necessary.
(f) Set reconstruction parameters and perform reconstruction. The filtered back projection (FBP) method was
used in our case.
(g) Export reconstructed result to Visualizer-kai. Perform
rendering to display the volume and sections of reconstructed 3D data (Fig. 7).
4 Notes
1. The lacey carbon film copper TEM grid is the most popular and
the cheapest grid to start with. The holes are variable which is
good for accommodating nonhomogeneous nanoparticles, but
the rigidity and stability of samples embedded in ice may not be
strong enough for some analysis due to the weak carbon support between holes. Extracontinuous carbon or formvar thin
film may therefore be needed for the enforcement of lacey
TEM grids.
2. The freshly prepared carbon films are hydrophilic, but they
become more hydrophobic over time. It is therefore necessary
to restore the surface hydrophilicity so that the aqueous solutions spread over the grid film easily and evenly. This can be
realized using plasma or glow discharging [9]. There are different types of glow dischargers on the market. But basically the
discharge current, pressure level, atmosphere gas and discharging time need to be tested to obtain the desired surface
property.
3. The successful application of immunogold labeling depends on
the capacity of antibodies to infiltrate throughout the lipid
membrane, and the specificity of recognition between
antigen-primary antibodies. The low antigen encapsulation in
archaeosomes also makes the labeling more challenging.
4. Labeling in a suspension method can be used. In this case,
primary Ab was added to an aliquot of archaeosomes in a vial
and incubated at 37
C for about 1 h. However, the subsequent
508
Hui Qian et al.