Biology Reference
In-Depth Information
stock under a variety of conditions is important not only for
laboratory work, but particularly for translational purposes that
require carefully controlled storage conditions. Fluorescence is
defined as the emission of light by a substance that has absorbed
electromagnetic radiation. Proteins contain three types of aromatic
amino acid residues (tryptophan, tyrosine, and phenylalanine) that
contribute to their intrinsic fluorescence. These residues produce
different fluorescence emission (both in wavelength and intensity)
depending on the polarity of the environment, with the final result
that the intrinsic fluorescence of proteins varies with their folding
state. Therefore, measurements of intrinsic fluorescence intensity
serve to probe perturbations in folding. However, in the case of a
large macromolecular complex such as adenovirus, intrinsic fluorescence
measurements are difficult to interpret. An alternative methodol-
ogy is the addition of dyes whose fluorescent properties reflect par-
ticular characteristics of the sample. This technique is known as
extrinsic fluorescence. Measurement of extrinsic fluorescence using
propidium iodide (PI) as a dye can be used to determine adenovi-
rus stability in a straightforward way. When intact, the viral capsid
is impermeable to PI. When the viral preparation is subject to
increasing stress levels (temperature, pH, ionic strength, etc.), at a
certain point the threshold for capsid structural integrity will be
exceeded and the capsid breaks, allowing access of PI to the
genome inside. PI binds to dsDNA by intercalating between the
bases, which causes a 20-30-fold enhancement of its fluorescent
emission. Therefore, monitoring PI fluorescence emission as stress
increases reveals the condition at which the capsid opens and
exposes the viral genome to the environment [
1
,
2
].
The rest of the chapter is devoted to visualization techniques
.
Three sections deal with different flavors of electron microscopy
(EM) aimed to image purified virus preparations; we then intro-
duce the use of atomic force microscopy (AFM) for imaging and
interacting with single virions without drying, in buffer conditions;
and finally, we describe two alternative techniques to prepare
infected cells for visualization at the electron microscope. Because
of the expensive equipment required, and the constant evolution
of the field, use of these techniques usually requires either access to
a state-of-the-art infrastructure facility, or collaboration with an
expert group.
The simplest preparation technique to examine virus morphol-
ogy by EM is
negative staining
[
3
]. Negative staining agents are
compounds of metals with high atomic number that serve to
scatter the electrons from regions covered with the stain, therefore
enhancing the contrast between the background and the biological
material in the image. The stain is excluded from the virus particle
and forms a cast around it, outlining its structure. It is also able to
penetrate between small surface projections to delineate them.
Areas where stain accumulates are dark in the image, while the
