Cryo electron microscopy

1. Cryo-Electron
Microscopy
THE NOBEL PRIZE IN CHEMISTRY,2017
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Presented by:
SOUMIT BOSE
3rd Semester
Instrumentation Science
Roll-04
Jadavpur University

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What is Microscopy?
Microscopy is the technical field of using microscopes to view object that cannot
be seen with the naked eye. There are three well known branches of microscopy:
1. Optical Microscopy
2. Electron Microscopy
3. Scanning probe Microscopy
Optical and electron microscopy involves the diffraction , reflection or refraction
of electromagnetic radiations or electron beam interacting with the specimen
Scanning Probe Microscopy involves the interaction of a scanning probe with the
surface of the object of interest.

3. Light Microscopy Electron Microscopy
Can study live
Color imaging
Relatively fast
cells Can study ultra-structure
Need to kill and ‘fix’ cells
Difficult and time consuming
Expensive
HIGH Resolution (upto 2 nm)
HIGH Magnification upto
100000x(SEM) and
250000x(TEM)
and easy
Relatively cheap
LOW Resolution (Limit
200 nm)
LOW Magnification upto
1000x
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How Do We Study Cells?

4. 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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5. Cryo-Electron Microscopy
A Form of TEM; sample is studied at Cryogenic
Temperatures

Native state of specimen is maintained ;
Specimens are observed in vitreous ice
Cryo-fixation; Rapid freezing of sample
Specimens are not stained or fixed

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Automated 3D image to get high resolution images

6. Origin and Development
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In 1975, Joachim Frank began work on the algorithms that would analyze
fuzzy 2D images and reconstruct them into sharp 3D structures.
In the early 1980s, Jacques Dubochet succeeded in vitrifying water, which
allowed the biomolecules to retain their shape even in high vacuum.
In 1990, Richard Henderson was the first to use an electron microscope to
generate a 3D image of a protein at atomic resolution.

7. Why Use It?
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Native state of sample is maintained – Cryo electron microscopy was meant to fight
radiation damage for biological specimens. The amount of radiation required to collect an
image of a specimen in the electron Microscope (EM) is high enough to damage delicate
structures of Specimen .In addition, the High vacuum required on the column of an
electron microscope makes the environment for the sample quite harsh.
Use of Vitreous ice to preserve biological sample – The biological sample is preserved by
Use of vitreous/amorphous ice. Common ice is a crystalline material where the molecules
are regularly arranged in a hexagonal lattice whereas amorphous ice is distinguished by a
lack of long-range order in its molecular arrangement. Amorphous ice is produced
by rapid cooling of liquid water (so the molecules do not have enough time to form a
crystal lattice) at low temperatures.
High resolution and Magnification Power- Resolution (~2nm) can be achieved with magnification
upto atomic resolution level .
3D Reconstruction can be achieved- Cryo electron tomography process( CET) is an imaging
technique used to produce high-resolution (~2 nm) three-dimensional views of samples, typically
biological Macromolecules and cells.

8. Schematic Diagram of Cryo-EM 8

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Procedures
Involved in Cryo-EM

10. 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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11. 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(b.p -195.79 c)
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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12. Cryo-Sectioning
Applicable for whole cells and tissues are too
thick to be spread 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 form.

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13. 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
Grids are:
A Carbon GridContinuous Films: Enable the sample to cover
the surface as a regular, thin layer.

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14. 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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15. 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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16. 3D Reconstruction
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3D images of biological sample is obtained by a process called
Cryo-Electron Tomography(CET),where a 3D reconstruction of a
sample is created from tilted 2D images.
Raw Images from different projections are recorded and at last
they are stitched together to form the required 3D structures
using computer software.

17. Images of sample
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18. Structure of Hepatitis B Virus Solved by
Cryo-EM
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Example

19. X RAY CRYSTALLOGRAPHY VS NUCLEUR MAGNETIC
RESONANCE (NMR)VS CRYO ELECTRON MICROSCOPE
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20. Pros and Cons
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ADVANTAGE : Structure remain in native state and no dehydration is
required. Thus the almost exact nature of water based molecules are
obtained.
Atomic resolution level (~2nm) is achieved.
Can be used for examining delicate sample structures .
One of the biggest advantages of cryo-electron microscopy is that it is
applicable for both larger and smaller specimens. Compared to other
microscopy techniques, cry-electron microscopy still produces good
images (as long as the sample is in good condition).
No requirement of staining or fixation of sample.
LIMITATION: It takes time to generate the sample.
Fully hydrated specimen have been shown to be electron-beam sensitive.
It is expensive .

21. 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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22. 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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ACKNOWLEDGEMENT
I am very grateful for the strong support and guidance
provided to me by Prof. Radhaballabh Bhar Sir who helped
me a lot in preparing this seminar
I am very thankful to him for same !

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