2. INTRODUCTION.
Use of cryo electron microscopy.
Precedures involved in microscopy.
Construction of the Technique
Applications.
Limitation.
3. Two types of the Electron Microscope:
Transmission Electron Microscope (TEM): A
beam of electrons interacts with the specimen
to form an image.
Scanning Electron Microscope (SEM): A beam
of electrons scans the sample surface to
create image.
4. Electrons can:
Pass through without interaction
Back or forward scattered, either elastically or
in- elastically.
Create secondary electrons and X-rays
Interact with Specimen.
5.
6. A Form of EM; sample is studied at Cryogenic
Temperatures.
It is not well defined at which point on the
temperature scale refrigeration ends and
cryogenics begin.
Native state of specimen; not stained not Fixed
Specimens are observed in vitreous ice.
Cryo-fixation; Rapid freezing of sample
Automated 3D image to get high resolution
images.
Low dose parameters are required so the sample
is not destroyed.
7. Native State of Sample
Vitrified Water
No Staining
Automated 3D Reconstruction
Validated Structures
8.
9.
10. A Cryo-EM is a TEM with an additional
specimen holder which:
Enable the viewing of the frozen-hydrated
specimen
Maintains Liquid Nitrogen or Liquid Helium
temperatures
11.
12. Two methods of specimen preparation are:
Thin Film: Specimen is placed on EM grid and
is rapidly frozen without crystallizing it.
Vitreous Sections: Larger samples are vitrified
by high pressure freezing, cut thinly and
placed on the EM grid.
13. Rapid Cooling is required to avoid the
formation of ice;
Rapid cooling traps the water in a vitrified
state in which it does not crystallize.
Vitrified state is maintained by keeping it at
liquid nitrogen temperature.
Vitrified state can be maintained for long
periods
Sample is placed on carbon grid and dipped
into a bath of ethane held in a container of
liquid nitrogen.
14. Whole cells and tissues are too thick to be
spread into a thin layer.
First vitrify sample and then cut into thin
sections using diamond knives.
Sectioning is a difficult task, distortions are
made in sample.
These distortions cause a loss in order of the
structure and makes it difficult for images to
increase the signal-to-noise ratio.
15. The grid on which the sample is placed is
made from carbon.
High quality carbon grid is used to get better
results.
Two types of Grids are:
Continuous Films: Enable the sample to cover
the surface as a regular, thin layer.
Holey Films: Have a network of holes of a
desired size in which the sample is spread.
16. Cryogens are used for Chilling and freezing
purpose
Type of cryogen used affects the rate of
freezing
Common cryogens are Liquid Nitrogen,
Ethane or Propane
Nitrogen is not directly used; It can make
crystals due to slow cooling.
17. The contrast of the specimen depends on:
Specimen itself
Defocus value of the objective lens
Thickness of the ice
There are three methods of observing and
recording images:
Fluorescent Screen.
Photographic Film.
CCD Cameras.
18. At low temperature and pressure, water
freezes into three forms:
Vitreous
Cubic
Hexagonal
Vitreous ice is obtained by rapid cooling of
liquid water.
19. 3D reconstruction process estimates the
unknown orientations and 3D structure at
the same time;
3D electron density maps are created from
2D projections
Angles of projections relative to each other
are determined
Find common line projections to determine
relative angles
20. 3D density map can be used to generate
projections that can be used to realign the
raw images.
Process may have to be repeated several
times.
21. Identify particles in micrograph and cut out
patches containing one particle each
This can be done automatically
Manual process is tedious and difficult
22. Image Noise is the random variation of
brightness or color information in images
produced by the sensor.
Cryo EM images are very noisy and have very
low contrast.
Smooth the noise as well as enhance the
contrast.
23.
24. Nanoparticle Research.
Pharmaceutical Drug Research.
3D Structure Visualization of:
Single Particles such as Ribosome, tRNA
Viruses
Proteins.
Macromolecules; Lipid Vesicles.
Used for 3D visualization of biological
molecules.
25. Very low to signal to noise ratio because the
biomolecules made up of carbon so image
contrast is low.
Sample must maintain at less than 135
degree Celsius.
Formation of virtuous liquid cannot be easily
formed.
Difficult to obtain images from frozen
sample.
