Lecture 1 & 2: Advances in microscopy: Electron microscopy (TEM, SEM) Flashcards
1
Q
Advances in microscopy: Electron microscopy (TEM, SEM)
A
TEM
2
Q
TEM
A
- basic approach (fixed tissue stained with uranyl acetate (uranium) & lead citrate). This remained unchanged for last 40 years.
- quality of micrograph depends on: speed of fixation (block size etc), nature of fixative used, thickness of section, quality of section & embedding
3
Q
Developments in TEM
A
- Cryomicrotomy (avoiding use of fixative)
- Negative staining
- High voltage instrument development
- Freeze fracture
- Specialist staining (including immunochemical methods)
- Digital imaging
- X-ray microanalysis (very important in materials science)
- Cryoelectron microscopy
4
Q
Cryoelectron microscopy
A
- used to elucidate structure of biomolecules at ultra-high R
- traditional TEM is unsuitable for such work (molecules degrade quickly)
- CryoEM is fast replacing X-ray crystallography for such work
5
Q
The rise of Cryo-electron microscopy
A
- taking over from x-ray crystallography as a method to deduce high-R protein structures, particularly of large molecules.
- x-ray crystallography: x-rays scatter as they pass through a crystallised protein; the resulting waves interfere with eachother, creating a diffraction pattern from which the position of atoms is deduced.
6
Q
Cryoelectron microscopy
A
- a beam of electron is fired at a frozen protein solution.
- the emerging scattered electrons pass through a lens to create a magnified image on the detector, from which their structure can be worked out
7
Q
Franks image analysis for 3D structures
A
8
Q
Dubochets vitrification method
A
9
Q
.
A
10
Q
Microscopy types - SEM
A
11
Q
SEM
A
- looks at surface structures
12
Q
SEM advantages
A
+ wide range of magnifications
+ good depth of field
+ large sample sizes
+ good resolutions (5nm)
