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Samenvatting hoorcollege 6
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Samenvatting hoorcollege 6 - Virusziekten uu
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Virusziekten samenvatting HC.6There are 3 types of structural biology determinating techniques:
1. X-ray crystallography
2. Nuclear magnetic resonance (NMR)
3. Electron microscopy (EM) focus of this lecture
Why use electrons for imaging? to be able to see smaller specimens (e.g. atoms).
Virusses are very small (range of 50-600 nm). With the human eye we can see up to 0.1 mm.
With light microscopes we can see up to 0.25 um (250 nm).
The resolving power (resolution) is the ability to separate points of an object that are located
at a small angular distance. The resolution is limited by the diffraction limit (d): the maximal
distance between two objects that the lens can resolve.
The wavelength of light is between 400-700 nm. When you take green light for example
(=500, NA=1), the diffraction limit is 250 nm. To image smaller objects, you need
something with a shorter wavelength.
One way to do that is to use
X-ray for imaging. You
make use of photons with a
lower wavelength.
X-ray properties:
High photon energy
Can penetrate thick samples
Small wavelength (10nm – 10pm)
Can ionize atoms and disrupt molecular bonds
Highly harmful for living tissues
No good lenses or mirrors!!
, Another thing that can be used are electrons. Unlike photons, electrons have a mass and
charge (affect way electrons interact with matter). But like photons, electrons show both
particle and wave characteristics.
The speed of light in a vacuum is constant but electrons can be
accelerated to high velocitys which results in short wavelengths. So the
velocity can be changes by changing the acceleration voltage.
Electrons are a type of ionization radiation. Unlike X-ray, there are lenses for electron beams
(beam of e can be easily redirected). EM provides atomic resolution information.
Sample preperation
Within the electron microscope (EM) electrons are
accelerates in high vacuum (needed) and have high
energy which is damaging for biological samples.
Biological samples considerations (EM):
Sample protection within the TEM e- are accelerated in high vacuum and have high
energy which is damaging for biological samples.
Fixation, staining, dehydration and plastic embedment
Sample thickness the sample needs to be thin enough for e- to go through (<1 um).
The penetration depth of e- increases with increasing acceleration voltage.
Thin film with thickness only slightly greater then the diameter of the specimen
Sectioning
Sample contrast biological samples mainly consist of light elements (H, C, N and O),
which weakly scatter e- and hence show poor contrast
Staining with heavy metals of high atomic number that are good electron scatterers
One problem is that dehydration alters the structure. The solution is keep the water and don’t
stain CryoEM.
Radiation damage is reduced at low temperatures. Cryo-electron microscopy (cryo-EM) is a
protection method that preserves the specimen structure in near-physiological condition
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