Cryo Electron Microscopy

1. Cryo-Electron
Microscopy
1

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
2

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
3

4. Light
Microscope
Electron
Microscope
4

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
5

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
6

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
7

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
8

9. Why Use It?
9
 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
11

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
12

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
13

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
15

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
16

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
17
A Carbon Grid

18. A Holey Grid
Supporting
Carbon Film
Ice Holes
Metal Grid
18

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
19

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
20
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
21

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
22

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
23

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
24

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
25

26. Structure of Hepatitis B Virus Solved by
Cryo-EM
26
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
27

28. Applications
 Nanoparticle Research
 Pharmaceutical Drug Research
 3D Structure Visualization of:
 Single Particles such as Ribosome, tRNA
 Viruses
 Proteins
 Macromolecules; Lipid Vesicles
28

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
29

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
30

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
31