Biology Reference
In-Depth Information
1. Place the mica incubated with the virus on the AFM holder.
2. Immerse the sample in 500 μL of 5 mM NiCl
2
in HBS buffer
(
see
Note 13
).
3. Place the holder with the mica on a piezoelectric actuator,
which controls the sample position.
4. Calibrate the cantilever spring constant (
see
Note 14
).
5. Pre-wet the AFM tip with 30 μL of NiCl
2
-HBS.
6. Focus the laser spot on the free extreme of the cantilever.
7. Tune the photodiode so that the laser spot is centered.
8. Proceed to bring the AFM tip closer to the surface of the
sample.
9. Turn on Jumping Mode plus (JM+) provided by WSxM (
see
Note 15
).
10. Optimize the measuring scanning parameters for best sample
imaging (
see
Note 16
). Scan large areas (about 3 × 3 μm) to
find viral particles on the surface.
11. Once a viral particle has been located, scan an area of
300 × 300 nm and 128 × 128 pixels to have enough resolution
to image a single viral particle (
see
Fig.
2g
) (
see
Note 17
).
12. If nanoindentation experiments are to be performed, obtain a
force-distance curve on a substrate area next to the viral parti-
cle. This will provide the cantilever deflection characteristics in
the absence of specimen.
13. Center on the viral particle and obtain a new force-distance
curve indenting on the particle beyond its rupture point (
see
Fig.
2h
).
14. Take a new image of the viral particle to observe the breakage
pattern (
see
Fig.
2i
).
15. Repeat for as many viral particles as needed to obtain adequate
statistical coverage.
3.5.2 AFM Setup
and Imaging
The AFM image processing software WSxM stores several data files
for the different features (topography, normal force, adhesion
among others) recorded with JM+. The following points must be
born in mind when analyzing the data:
3.5.3
Image Analysis
1. Topography images usually present an artifactual tilt when the
surface is not totally perpendicular to the z-scan. To eliminate
this effect, computationally fit the surface with a plane from an
area free from viral particles.
2. Changing the color scale and equalizing the data helps reveal
details of the virion topography.
