Biology Reference
In-Depth Information
excluded areas (containing the biological material) are light—there-
fore the “negative” in the technique's name. Negative staining is a
fast technique that requires small sample amounts. In a large and
faceted icosahedral particle like the adenovirus virion, individual
capsomers can be discerned using this technique. In fact, adenovi-
rus was one of the first viruses to be imaged at the electron micro-
scope [
4
].
The purely morphological information provided by negative
staining can be complemented with labeling techniques to detect
specific components in the sample. Labeling is particularly relevant
when imaging a sample with complex protein composition such as
adenovirus. The
negative staining immunoelectron microscopy
tech-
nique [
5
] can be used to detect the presence, location, and acces-
sibility of proteins or peptides in the virion. In this technique, the
virus is adsorbed onto a carbon support film, followed by a series
of “on grid” labeling and washing steps and a final negative stain-
ing. The bound antibody is visualized using a secondary antibody
conjugated with gold particles. After negative staining, the gold
particles show up in the microscope as black dots.
Negative staining is a straightforward, extremely valuable sam-
ple preparation method for EM visualization of viruses. However,
it must always be considered that the specimen is observed in con-
ditions quite different from its physiological state: staining agents
may have extreme pH values; the sample is dried; and the image
represents the cast left by the embedding heavy metal, while the
actual biological material is destroyed during imaging due to the
high vacuum and radiation conditions in the microscope.
Cryo-
electron microscopy
(cryo-EM) allows visualization of samples in a
more close-to-native environment [
6
]. For cryo-EM, a thin layer
(~1,000 Å) of sample preparation in an appropriate buffer solution
is frozen by immersion in a cryogen (e.g., liquid ethane), at a speed
high enough to prevent formation of ice crystals that would destroy
the specimen structure [
7
]. The sample, in this frozen hydrated
state, is imaged at low temperatures (ca. −180 °C) [
8
]. Unlike
negative staining, cryo-EM images contain information directly
related to the specimen density at each point, including the interior
of viral particles. A disadvantage of cryo-EM is that images have
very low signal to noise ratio, due to the lack of contrasting agent
and to the use of very low electron doses in image acquisition to
prevent radiation damage. As a consequence, cryo-EM images are
often hard to interpret in a direct way. Both negative staining and
cryo-EM provide projection images, merging in a single plane the
information coming from the different heights in the object.
Three-dimensional (3D) information can be recovered using
sophisticated image processing methods [
9
,
10
]. Adenovirus was
also one of the first specimens to be studied in 3D using cryo-EM
[
11
]. Technical improvements in the past years resulted in the
recent solution of the virion structure at 3.5 Å resolution, similar
