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cryo electron microscopy
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- 5. Content 5 Introductionto ElectronMicroscopy WhyUseCryo-EM? ProceduresInvolvedin Cryo-EM ProsandConsoftheTechnique Applications FutureProspects Conclusion
- 6. DEFINITIONS: ”Cryoelectron microscopy isa method for imaging frozen-hydrated specimens at cryogenic temperatures by electronmicroscopy”
- 7. MEANING Cryo: “A combining formmeaning 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 WeStudy Cells? 8 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
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- 10. Electron Microscopy 1 0 Twotypesof theElectronMicroscope: TransmissionElectronMicroscope(TEM):Abeam of electrons interacts with the specimento form an image ScanningElectronMicroscope(SEM): Abeamof electrons scansthe samplesurface to create image
- 11. I nterac tion 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 1 1
- 12. Cryo-Electron Microscopy 1 2 A FormofEM;sampleisstudied at CryogenicTemperatures Native state of specimen;not stainednot Fixed Specimensare observedin vitreousice Cryo-fixation; Rapidfreezing of sample
- 13. Cont.. • Automated 3Dimagetoget high resolutionimages • Lowdoseparameters are required sothe sample isnot destroyed
- 14. Origin and Development 1 4 1980s BruggellerandMayer 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 IT ? 1 6 Native State ofSample Vitrified Water NoStaining Automated 3DReconstruction ValidatedStructures
- 17. 10Schemat i cDi agr am of Cryo-EM
- 18. Procedures Involv ed inCryo-EM 18
- 19. The Microscope ACryo-EM isaTEMwithan additional specimenholder which: Enablethe viewing of the frozen-hydrated specimen Maintains Liquid Nitrogen or Liquid Helium temperatures 19
- 20. Specimen Preparation Twomethods ofspecimenpreparationare: ThinFilm:Specimenisplaced on EMgrid and is rapidly frozen without crystallizingit VitreousSections:Largersamplesare vitrified by high pressurefreezing, cut thinly andplaced on the EMgrid 20
- 21. Adrop of thesampleisplaced at the endof the plungerandrapidly immersedinto the cryogentank 21
- 22. 1) Vitrification RapidCooling isrequired 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 22
- 23. Cont.. • Vitrified statecanbe 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 bespreadintoathinlayer 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 ids 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 25
- 26. A Holey Grid Supporting CarbonFilm Ice Holes Metal Grid 26
- 27. Cryogens 27 Cryogensare usedfor Chillingand freezingpurpose Typeof cryogenusedaffects the rate offreezing Commoncryogensare LiquidNitrogen,Ethaneor Propane Nitrogen isnot directly used; It canmakecrystalsdue to slow cooling
- 28. Formation of ic 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 28
- 29. Observation of theSpecimen 29 Thecontrastof the specimendependson: Specimen itself Defocusvalue of the objectivelens Thicknessof theice
- 30. 3D Reconstruction 30 3Dreconstructionprocessestimates theunknown 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 densitymap can be used to generate projections that can be used to realign the raw images •Processmay have to be repeated severaltimes 31
- 32. • Thereare threemethodsof observingandrecording images: • FluorescentScreen • PhotographicFilm • COD Cameras METHODS
- 34. 2) Automated ParticlePicking Identify particles in micrograph and cut out patchescontaining one particleeach Thiscanbe doneautomatically Manual processistedious anddifficult 34
- 35. 3) Images Enhancement ImageNoise 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 35
- 36. Structure of HepatitisB Virus Solved by Cryo-EM Example 36
- 37. Pros and Cons 37 Advantage:Structureremains 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 tions 38 Nanoparticle Research Pharmaceutical DrugResearch
- 39. CONT.. • 3DStructure Visualizationof: • SingleParticles suchasRibosome, • Viruses • Proteins • Macromolecules; LipidVesicles
- 40. Future Prospects 40 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 42 A. Cryo-EM isa 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 ofCryo-EM is not high enough but it is improving using different computertechniques • With the advancement of technology, this technique will certainly improve
- 44. REFERENCES 44 • 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.