Biology Reference
In-Depth Information
mica (provided that bivalent cations are present in the buffer
solution) or functionalized glass.
6. The total volume of sample depends on the particular spectro-
fluorimeter and cuvette models used. The requirement is that
the sample lies in the optical path.
7. Double distilled water can also be used for washing, but it may
be damaging to the structures due to osmotic shock, and may
induce staining artifacts (e.g., positive staining).
8. A common problem in negative staining is its variability: even
within the same grid square, negative and positive staining
areas may be observed. Positive staining arises when uranyl
acetate binds to the biological material, instead of being
excluded. In positively stained areas, viral particles appear dark
on a light background, and structural details are no longer vis-
ible (
see
Fig.
2c
). These areas must not be used for structural
studies. Uranyl acetate usually works well for adenovirus, but if
needed, alternative staining agents such as ammonium molyb-
date can be used. Ammonium molybdate is usually prepared as
a 2 % w/v solution in double-distilled water (pH ~ 5.3) and it
can be titrated with NaOH or NH
4
OH to pH 6.0-7.0.
9. The virus sample should appear homogeneously spread all over
the grid, allowing observation of isolated particles. An optimal
virus concentration would be 1 × 10
11
vp/mL.
10. TBS/BSA and TBG can be stored in aliquots at −20 °C. TBG
has to be thawed at 37 °C to melt the gelatin.
11. It is crucial that any tool, storage element, etc. that will be in
contact with the grid after vitrification is precooled in liquid
nitrogen. Otherwise the sample temperature will rise and
undesirable ice crystal formation, or even thawing, will occur.
12. As an alternative to averaging, cryo-electron tomography
allows recovery of the 3D structure of individual viral particles
without averaging, providing information on unique structural
features [
30
]. For cryo-electron tomography, the viral sample
is mixed with 10-nm colloidal gold particles (AURION,
Wageningen, The Netherlands) before plunge freezing. The
electron microscope must be equipped with a high-tilt cryo
holder and an automated image acquisition system [
31
], to
take a tilt series of the same field covering an angular range
between −70 and +70 °C. The images in the tilt series are
aligned using the gold particles as fiducials, normalized and
reconstructed using specialized software packages [
32
-
34
].
13. A large volume of buffer reduces the risk of high variations of
ionic strength due to evaporation, ensuring virus stability
throughout the experiment.
14. Among the available methods [
35
], the one proposed by Sader
[
36
] works very well for the cantilevers used here.
