cryoelectron microscopy

3. Khurshid anwar
BS BOTANY 8TH

4. Cryo- ELECTRON
Microscopy
4

5. Content
5
Introductionto ElectronMicroscopy
WhyUseCryo-EM?
ProceduresInvolvedin Cryo-EM
ProsandConsof theTechnique
Applications
FutureProspects
Conclusion

6. DEFINITIONS:
”Cryoelectron microscopy is a method for imaging
frozen-hydrated specimens at cryogenic
temperatures by electronmicroscopy”

7. MEANING
Cryo:
“A combining form meaning icy cold, frost”
Microscopy:
“Technical field of using microscopes to view objects
and areas of objects that cannot be seen with the
naked eye”

8. How Do We Study Cells?
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Can study live cells
Colorimaging
Relatively fast andeasy
Relativelycheap
Resolution Limit 200nm
Light Microscopy Electron Miic roscopy
Can study ultra-structure
Needto kill and ‘fix’ cells
Difficult and time
Consuming Expensive
Resolution Limit 2nm

9. Light
Microscope
Electron
Microscope
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10. Electron Microscopy
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Twotypesof the ElectronMicroscope:
TransmissionElectronMicroscope(TEM):Abeam of
electrons interacts with the specimento form an
image
ScanningElectronMicroscope(SEM):
Abeamof electrons scansthe samplesurface to create
image

11. I nterac t ion of Elect onswith Specimen
 Electronscan:




Pass through without
interaction
Back or forward scattered,
either elastically or in-
elastically
Create secondary electrons
andX-rays
Interact with Specimen
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12. Cryo-Electron Microscopy
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A Formof EM;sampleisstudied at CryogenicTemperatures
Native state of specimen;not stainednot Fixed
Specimensare observedin vitreousice
Cryo-fixation;
Rapidfreezing of sample

13. Cont..
• Automated 3Dimageto get high resolutionimages
• Lowdoseparameters are required sothe sample isnot
destroyed

14. Origin and Development
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4
1980s
Bruggellerand Mayer produced vitrified water
Dubochet and McDowall produced a thin layer of vitrified
waterusing liquid ethane
All these findings have been combined to produce a simple
but extremely powerful method for the production of high-
resolution images

16. WHy Use I T ?
1
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Native State ofSample
Vitrified
Water
NoStaining
Automated 3DReconstruction
ValidatedStructures

17. 10Schemat i c Di agr am of Cryo-EM

18. Procedures
Involv ed in Cryo-EM
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19. The Microscope
ACryo-EM isaTEMwith an
additional specimenholder
which:
Enablethe viewing of the
frozen-hydrated specimen
Maintains Liquid Nitrogen
or Liquid Helium
temperatures
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20. Specimen Preparation
Twomethods of specimenpreparationare:
ThinFilm:Specimenisplaced on EMgrid and is
rapidly frozen without crystallizingit
VitreousSections:Largersamplesare vitrified by
high pressurefreezing, cut thinly andplaced on
the EMgrid
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21. Adrop of the sampleisplaced at the endof the plungerandrapidly
immersedinto the cryogentank
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22. 1) Vitrification
RapidCooling is required to
avoidthe formation of ice:
Rapidcooling traps the water ina
vitrified state in which it doesnot
crystallize
Vitrified state ismaintained by keeping
itat liquid nitrogentemperature
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23. Cont..
• Vitrified state canbe maintained forlong periods
Sample is placed on carbon grid and dipped into a bath
of ethane held in acontainer of liquid nitrogen

24. 2) Cryo-Sectioning
2
4
Wholecellsandtissuesaretoothickto bespreadintoathin layer
Firstvitrify sampleandthen cut into thin sectionsusingdiamond knives
Sectioningisadifficult task,distortions are madeinsample
Thesedistortions causealossin order of the structure and makesit
difficult for imagesto increasethe signal-to-noiseratio

25. Cryo-EM Gr i ds
The grid on which the sample is placed is made from
carbon
Highquality carbongrid isusedto get better results
Twotypes of Gridsare:
Continuous Films: Enable the sample to cover
the surface asaregular, thin layer
Holey Films: Have a network of holes of a
desired sizein which the sampleisspread
ACarbonGrid
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26. A Holey Grid
Supporting
Carbon Film
Ice Holes
Metal Grid
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27. Cryogens
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Cryogensare usedfor Chilling and freezingpurpose
Typeof cryogenusedaffects the rate offreezing
Commoncryogensare LiquidNitrogen,Ethaneor Propane
Nitrogen isnot directly used; It canmakecrystalsdue to slow
cooling

28. Formation of i c e
At low temperature andpressure,
water freezes into threeforms:
Vitreous
Cubic
Hexagonal
Vitreous ice isobtained byrapid
cooling of liquidwater
An Ice Hole
Particles are randomly
positioned and
orientated
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29. Observation of the Specimen
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Thecontrastof the specimendependson:
Specimen itself
Defocusvalue of the objectivelens
Thicknessof theice

30. 3D Reconstruction
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3Dreconstructionprocessestimates the unknown
orientations and 3Dstructure at the sametime;
3Delectron density mapsare created from 2Dprojections
Anglesof projections relative toeachother are determined
Findcommon line projections todetermine relative angles

31. 1) Re-projections
•3D density map can be used to generate
projections that can be used to realign the raw
images
•Processmay have to be repeated severaltimes
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32. • Thereare three methodsof observingandrecording
images:
• FluorescentScreen
• PhotographicFilm
• COD Cameras
METHODS

34. 2) Automated Particle Picking
Identify particles in micrograph and cut
out patchescontaining one particleeach
Thiscanbe doneautomatically
Manual processistedious anddifficult
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35. 3) Images Enhancement
Image Noise is the random variation of brightness or color
information in imagesproduced by thesensor
Cryo EMimagesare very noisy and havevery lowcontrast
Smooth the noise aswell asenhancethe contrast
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36. Structure of Hepatitis B Virus Solved by
Cryo-EM
Example
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37. Pros and Cons
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Advantage:Structure remains native andno
dehydration isrequired
Limitation: It is not possible to look at the sample for a
long time because of beam damage and it causes poor
resolution

38. Applic a t ions
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Nanoparticle Research
Pharmaceutical DrugResearch

39. CONT..
• 3DStructure Visualization of:
• SingleParticles suchasRibosome,
• Viruses
• Proteins
• Macromolecules; LipidVesicles

40. Future Prospects
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Anumberof improvementsareexpectedinfuture
• HighVoltage ElectronSource
•Mathematical Correction of Lens
• DefectsHighResolution

41. Cont..
• Improved Scanners
• Better High-PressureFreezing
• Improved CODdetectors will remove the need for
computer
• processing in future

42. Conclusions
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A. Cryo-EM is a form of Transmission Electron
Microscopy (TEM) where the sample is studied in its
native state at cryogenic temperatures
• Usedfor 3Dvisualization of biologicalmolecules

43. Cont..
• Resolution of Cryo-EM is not high enough but it is improving
using different computertechniques
• With the advancement of technology, this technique will
certainly improve

44. REFERENCES
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• Cryo-electron microscopy, Methods in Molecular Biophysics,
Spring2009
• Cryo-electron microscopy: taking back the knight
Stephen Fullerpy, MICROBIOLOGYTODAY VOL
26/MAY 99
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678009/pd
f/nihms87373.pdf
• http://www.bbc.co.uk/dna/hub/A914302#back10
• http://www.physorg.com/news192189631.html

45. C0NT..
• http://en.wikipedia.org/wiki/Cryo-
electron_microscopy
• http://www.jic.ac.uk/microscopy/intro_EM.html
• http://en.wikibooks.org/wiki/Structural_Bi
ochemistry/Proteins/CryoElectron_Microscpy
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726
835/pdf/nihms100338.