Biology Reference
In-Depth Information
4. Transfer the grid from the ethane to a precooled cryo-box and
store in liquid nitrogen until used. Make sure that once vitri-
fied, the grid is never exposed to air or temperatures higher
than −160 °C. Otherwise, undesirable effects due to tempera-
ture increase (
see
Note 11
) or contamination by condensation
may occur.
5. Cool down the electron microscope anticontaminator and the
cryo-sample holder using a cryo-transfer device and liquid
nitrogen. When the cryo-holder reaches a stable temperature
below −170 °C, transfer the grid from the cryo-box to the
sample holder using precooled tweezers.
6. Load the cryo-holder on the electron microscope. Wait for
30 min while monitoring that the holder temperature stays
stable below −170 °C.
7. Observe the grid at low magnification (950×). Select grid squares
with an adequate ice thickness and record their positions.
8. Set up the microscope to low dose imaging parameters, so that
the sample areas of interest are irradiated exclusively during
image recording while focusing is performed in an adjacent
location.
9. Record micrographs at 60,000× magnification in low dose
conditions, covering a defocus range between −1.5 and
−3.5 μm (
see
Fig.
2f
).
10. Digital format micrographs (either directly recorded if the
microscope is equipped with a digital image acquisition sys-
tem, or digitized in a high quality scanner such as the Nikon
Coolscan 9000) are processed for viral particle extraction,
alignment, and combination into a 3D reconstruction. The
resolution attained in the final map will be related not only to
the number of particles averaged but also to the quality of each
individual image. A variety of specialized image processing
software tools are available in the field [
25
]. Some popular
ones for reconstruction of icosahedral virus particles are
FREEALIGN [
26
], XMIPP [
27
], Bsoft [
28
], or IMIRIS [
29
]
(
see
Note 12
).
3.5 Atomic Force
Microscopy
1. Cut a ~1 cm
2
piece from a mica sheet.
2. Cleave mica with tape to obtain a clean surface.
3. Dilute the adenovirus sample from a single-use aliquot in the
solution of NiCl
2
in HBS to obtain a final solution of
~5 × 10
11
vp/mL and 5 mM Ni
2+
.
4. Place a 20 μL drop of final solution on the mica substrate.
5. Incubate for 30 min at 4 °C.
6. Wash the sample with a solution of 5 mM NiCl
2
in HBS.
3.5.1 Sample
Preparation
