Cryo electron microscopy

Cryo Electron Microscopy

1. Cryo-Electron
Microscopy
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2. Overview
Introduction to Electron Microscopy
Why Use Cryo-EM?
Procedures Involved in Cryo-EM
Pros and Cons of the Technique
Applications
Future Prospects
Conclusion
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3. How Do We Study Cells?
Light Microscopy
Can study live cells
Color imaging
Relatively fast and easy
Relatively cheap
Resolution Limit 200 nm
Electron Microscopy
Can study ultra-structure
Need to kill and ‘fix’ cells
Difficult and time consuming
 Expensive
Resolution Limit 2 nm
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4. Light
Microscope
Electron
Microscope
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5. Electron Microscopy
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
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6. Interaction of Electrons With
Specimen
Electrons can:
Pass through without
interaction
Back or forward scattered,
either elastically or in-
elastically
Create secondary electrons
and X-rays
Interact with Specimen
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7. Cryo-Electron Microscopy
A Form of EM; sample is studied at Cryogenic Temperatures
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
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8. Origin and Development
1980s:
 Bruggeller and Mayer produced vitrified water
Dubochet and McDowall produced a thin layer of vitrified water using
liquid ethane
All these findings have been combined to produce a simple but
extremely powerful method for the production of high-resolution
images
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9. Why Use It?
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Native State of Sample
Vitrified Water
No Staining
Automated 3D Reconstruction
Validated Structures

10. 10Schematic Diagram of Cryo-EM

11. Procedures
Involved in Cryo-EM
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12. The Microscope
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
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13. Specimen Preparation
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
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14. 14
A drop of the sample is placed at the end of the plunger and rapidly
immersed into the cryogen tank

15. 1) Vitrification
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
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16. 2) Cryo-Sectioning
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
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17. Cryo-EM Grids
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
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A Carbon Grid

18. A Holey Grid
Supporting
Carbon Film
Ice Holes
Metal Grid
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19. Cryogens
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
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20. Formation of Ice
At low temperature and pressure,
water freezes into three forms:
 Vitreous
 Cubic
Hexagonal
 Vitreous ice is obtained by rapid
cooling of liquid water
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An Ice Hole
Particles are randomly
positioned and
orientated

21. Observation of the Specimen
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
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22. 3D Reconstruction
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
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23. 1) Re-projections
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
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24. 2) Automated Particle Picking
Identify particles in micrograph and cut
out patches containing one particle each
This can be done automatically
Manual process is tedious and difficult
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25. 3) Images Enhancement
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
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26. Structure of Hepatitis B Virus Solved by
Cryo-EM
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Example

27. Pros and Cons
Advantage: Structure remains native and no
dehydration is required
Limitation: It is not possible to look at the sample for a
long time because of beam damage and it causes poor
resolution
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28. Applications
Nanoparticle Research
Pharmaceutical Drug Research
3D Structure Visualization of:
Single Particles such as Ribosome, tRNA
Viruses
Proteins
Macromolecules; Lipid Vesicles
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29. Future Prospects
A number of improvements are expected in future
High Voltage Electron Source
Mathematical Correction of Lens Defects
High Resolution
Improved Scanners
Better High-Pressure Freezing
Improved CCD detectors will remove the need for computer
processing in future
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30. Conclusions
Cryo-EM is a form of Transmission Electron Microscopy (TEM)
where the sample is studied in its native state at cryogenic
temperatures
Used for 3D visualization of biological molecules
Resolution of Cryo-EM is not high enough but it is improving using
different computer techniques
With the advancement of technology, this technique will certainly
improve
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31. Literature
Cryo-electron microscopy, Methods in Molecular Biophysics, Spring 2009
Cryo-electron microscopy: taking back the knight Stephen Fullerpy,
MICROBIOLOGYTODAY VOL 26/MAY 99
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678009/pdf/nihms87373.pdf
http://www.bbc.co.uk/dna/hub/A914302#back10
http://www.physorg.com/news192189631.html
http://en.wikipedia.org/wiki/Cryo-electron_microscopy
http://www.jic.ac.uk/microscopy/intro_EM.html
http://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Cryo-
Electron_Microscopy
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726835/pdf/nihms100338.pdf
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