Biology Reference
In-Depth Information
to that obtained by protein crystallography [
12
,
13
]. Cryo-EM
studies have also revealed details on adenovirus receptor or anti-
body binding (
reviewed in
[
14
]), as well as uncoating [
2
].
The most novel technique we address here is
Atomic Force
Microscopy
(AFM) [
15
]. AFM was first described in 1986, and has
been used in different research fields since then, most notably in
materials science. The atomic force microscope belongs to the
scanning probe microscope (SPM) family. It consists of a sharp tip
at the end of a cantilever, which bends depending on the interac-
tions between the tip and the sample. The tip sharpness and the
scanning conditions play an important role in the AFM resolution,
which under the appropriate conditions can even reach atomic
level [
16
]. AFM has become a widely used tool in biophysics, due
to its ability to apply forces as small as piconewtons (pN) and
resolve structures in the μm to nm range [
17
]. AFM provides
information on the topography of the sample surfaces that are
accessible to the tip, but not on internal features. Apart from pro-
viding this purely visual information, the specific potential of AFM
lies in its ability to interact with the specimen and measure forces
at single molecule resolution. AFM allows to measure physical
properties such as stiffness or surface charge density, among others
[
18
-
20
]. In physical virology, AFM is helping to relate structure to
physical properties in single viral particles [
2
,
21
-
23
].
All the techniques mentioned so far refer to analyses of purified
virus particles. When
visualization of infected cells
at the electron
microscope is desired, specific preparation techniques must be used
to allow structural preservation, sectioning at the required thick-
ness (~70 nm) and contrast generation. Different variations of
chemical cross-linking, resin embedding, and staining can be used
depending on the particular experimental goal. Here we describe a
standard procedure for room temperature embedding, aimed at
providing high contrast and structural preservation for morpho-
logical characterization. We also indicate a variant of the technique
consisting in a milder, low temperature embedding procedure,
providing conditions in which immunogenic properties are pre-
served to allow labeling on cell sections.
2
Materials
1. Sealed quartz cuvette with a light path of 10 mm, and volume
of 700 μL (Hellma
®
absorption cuvette or similar).
2. Spectrofluorimeter equipped with filters for excitation at
353 nm and emission monitoring from 550 nm to 650 nm and
a Peltier cooler (Hitachi Model F-2500 FL or equivalent).
3. Purified adenovirus sample at 5 × 10
10
viral particles/mL
(vp/mL) concentration.
2.1 Fluorescence
Spectroscopy
