Cryogenic electron microscopy is a technique that uses electron microscopy to image samples that have been rapidly frozen to preserve their structure. The presentation discusses the principles, procedures, and applications of cryo-EM. It explains that samples are vitrified to prevent ice crystal formation before being imaged with an electron microscope. This allows structures like proteins to be viewed in their native state at high resolution. The presentation outlines the sample preparation, imaging, and image enhancement processes and discusses how cryo-EM is being used to determine the structures of biological molecules and systems. It predicts that cryo-EM will continue advancing to study even larger complexes and become more accessible and useful for research.

1. PRESENTATION BY
ALIZAH ARSHAD
ESHMAL JOHN
IQRA

2. CRYO ELECTRON MICROSCOPY

3. INTRODUCTION
• Cryogenic electron microscopy is a cryomicroscopy technique applied
on samples cooled to cryogenic temperatures.
• In cryo-EM, samples are rapidly frozen (vitrified), preventing the
formation of crystalline ice and preserving samples in their natural
state. A transmission electron microscope (TEM) is then used to
image the sample, capturing a two-dimensional view, or projection, of
the specimen

4. RESOLUTION OF CRYO ELECTRON MICROSCOPY

5. PRINCIPLE OF cryo-EM
• For biological specimens, the structure is preserved by embedding in
an environment of vitreous ice.
This technique is mainly aimed at the development of new approaches
to enhance the image resolution of protein structures within cellular
conditions.

6. PROCEDURE
• The sample must be vitrified.
• Strong beam of electrons is used.
• The grid on which sample is placed is made up of carbon.
• Cryogens are used for freezing purposes.
types of cryogens are used
• Common cryogen are liquid nitrogen, ethane or propane.

7. SAMPLE PREPARATION
• The sample is prepared by two methods
• Thin film:
• The sample is place on grid and is frozen without crystallization
• Vitreous section :
• The sample is placed on grid and is vitrified and thinly cut .

8. VITRIFICATION
• The grid preparation process, often referred to as vitrification, is conceptually
simple to understand: the aqueous sample is applied to a grid, the sample is
made thin on the grid, and then the grid is plunge frozen at a time scale that
prevents the formation of crystalline ice.
• Vitrified state can be maintain for long time
• Purified samples are vitrified by plunging in liquid ethane cooled by liquid
nitrogen.
• liquid ethane has higher heat transfer capacity and results in better vitrification

9. CRYO SECTIONING
• Cryo-electron microscopy of vitreous sections is, in principle, the ultimate
method of specimen preparation. It consists in ultra-rapid cooling of a sizable
sample of biological material that is cut into thin sections.
• vitrified sample are cut into thin sections using diamond knife.

10. INTERACTION OF ELECTRON WITH SPECIMEN
• It works by flash-freezing biological samples in glass-like ice and using a high-
energy electron beam to probe the specimen. Upon firing the electrons at the
sample, the atoms within the sample scatter and change their direction.
• Back or forward scattered of electrons .
• Once the beam hits the sample, electrons and X-rays are ejected from the
sample.

11. OBSERVATIONS
• Observations is made on the basis of
a. Specimen itself
b. Thickness of ice
c. Focus of objective lenses
• Methods of observation
a) Fluorescent screen
b) Photographic film

12. IMAGE ENHANCEMENT
• Cryo EM image are very noisy and have very low contrast
• Smooth the noise as well as enhance the contrast.
• Color information and brightness is produced by sensor

13. IMAGE ENHANCEMENT

14. PROS AND CONS
• Pros:
a) Easy sample preparation
b) Structure in native state
c) Small sample size
d) Précised image
• Cons:
a) Relatively low resolution
b) Highly dependent on EM techniques
c) Costly EM equipment

15. APPLICATIONS
• Electron microscopy (EM) is a technique for obtaining high resolution images of
biological and non-biological specimens. It is used in biomedical research to
investigate the detailed structure of tissues, cells, organelles and macromolecular
complexes.
• Determining the structure of proteins and other macromolecules: Cryo-EM can
be used to determine the three-dimensional structure of proteins and other
macromolecules at high resolution. This information can be used to understand
how these molecules function and how they interact with other molecules.
• Studying complex biological systems: Cryo-EM can be used to study complex
biological systems such as viruses, membrane proteins, and multi-protein
complexes. It can provide insights into the mechanisms of these systems and how
they interact with their environment

16. FUTURE PROSPECTS
• The future prospects for cryo-electron microscopy (cryo-EM) are very promising.
• As cryo-EM technology continues to improve, it is likely that we will be able to
obtain even higher resolution images of even larger and more complex biological
molecules. This will allow us to better understand the structure and function of
these molecules, which will have important implications for drug discovery,
biotechnology, and other areas of research.

17. FUTURE PROSPECTS
• In addition, cryo-EM is becoming more accessible to researchers around the
world as the technology becomes more affordable and user-friendly. This will
allow more scientists to use cryo-EM in their research, leading to even more
discoveries and breakthroughs in the coming years

18. CONCLUSION
• To summarize, cryo-EM has emerged as one of the effective imaging
techniques to determine the structures of biological assemblies and
macromolecules. In the future, cryo-EM can be potentially used for
structure-based drug discovery.

19. THANKS