Presentation on theme: "ELECTRON MICROSCOPY Introduction Principles of operation of TEM"— Presentation transcript:
1 ELECTRON MICROSCOPY Introduction Principles of operation of TEM Sample preparation for TEMAdvantages and disadvantages of TEMPrinciples of operation of SEMSample preparation for SEMAdvantages and disadvantages of SEMSchool of Biological SciencesInvestigating micrographs
2 INTRODUCTIONWith the invention of the light microscope it was discoveredthat plant and animal tissues were made up of aggregates ofindividual cells. However, light microscopes are limited toapproximately x1000 magnification and have poor resolution.Therefore not all the internal structures of a cell can be seenwith a light microscope.In 1924 a French physicist by the name of de Broglie stated that a beam of electrons should behave in a similar way to a beam of light i.e with wave properties the wavelength should be shorter. Therefore an electron beam should give better resolution.
3 RESOLUTIONWhen there is sufficient light, two points 0.2mm apart or morecan be distinguished with the naked eye as being separatepoints. When this distance is less than 0.2mm, only one point isseen. This distance is called the resolving power (or resolution)of the eye.In other words resolution is the closeness two objects can be inproximity and still be perceived as two separate objects.Can’t see two separate objectsCan see two separate objects
4 The invention of the electron gun led to the development of theelectron microscope.The metal tungsten filament isheated to about 2500oC whichcauses it to release electrons.Due to the large voltagedifference between thefilament and the anode platethe electrons are forced toflow in the direction of thearrow.The cathode shield increasesthe electron flow further andconcentrates the electrons intoa narrow beam.Back to principles of operationFilamentCathodeHigh voltage generatorElectron beamAnode
5 Two main types of electron microscopes; TEM TransmissionSEM ScanningTEM produces a high resolution image of the internal structures of cells. TEM uses the electrons that have passed through the specimen to form an image.SEM produces a three dimensional image of the specimen surface. A beam of electrons scans the whole specimen which then emits low energy, secondary electrons. This technique can be used to study whole cells.
6 When an electron beam strikes a specimen a number of events occur When an electron beam strikes a specimen a number of events occur. Electrons are scattered depending on the nature of the material.If the electrons hit a dense array they are scattered out of the main beam and fewer electrons will reach the viewing screen. There is no fluorescence and that area appears dark.If the electrons pass a scarcity of atoms they travel straight through, hitting the viewing screen and causing fluorescence. That area will appear light. The image comes from the arrangement of light and dark patches on the screen.
7 PRINCIPLES OF OPERATION OF TEM Electron gunSpecimen holderProjection chamber
8 The main components of a TEM are: THE ELECTRON GUN – producesan electron beam.THE COLUMN – uses electromagneticlenses to control the beam andproduce a magnified imageIMAGE VIEWING AND RECORDINGThe image is produced on afluorescent screen below which ashutter and camera are located.eeCondenser lenseeSpecimeneObjective lenseeeProjector lenseee e ee e eee e eeeeVacuumFluorescent screen
9 Electrons only behave like light when they are manipulated in vacuum Electrons only behave like light when they are manipulated in vacuum. Therefore the whole column is evacuated since atoms such as O2 and CO2 scatter the electrons.e eee ee e
10 SAMPLE PREPARATION FOR TEM The aims of sample preparation are as follows:To preserve the material in its natural stateTo ensure that the material withstands changes which might occur on exposure to atmosphere, vacuum and electron beam.
11 Fixation for TEM It is then placed into fixing solution The tissue is cut into tiny piecesIt is then placed into fixing solution
12 Dehydration and embedding of TEM Tissue is placed in final embedding mixture and the resin is polymerised in the ovenIt is then placed in a dilute solution of resin embedding mediaTissue is dehydrated in alcoholSpecimen vials
13 Section cutting of TEMSections are cut on an ultramicrotome with a glass or diamond knife. The sections are floated off the edge of the knife onto the surface of a water trough.The colour of the sections vary with thickness. When the sections are gold they are picked off the surface with a copper grid.The section on the copper grid is now ready for staining and viewing in the electron microscope.3.05mm
14 Advantages Disadvantages TEMVery good resolutionThin sections are effectively two dimensional slices of tissue and do not convey the three dimensional arrangement of cellular componentsmagnificationArtefacts may be createdCan see sub-cellular components and measure them
15 PRINCIPLES OF OPERATION OF SEM Electron gunDetectorDetectorImage viewingSpecimen chamberControl panel
16 SEM uses electrons that are emitted from the specimen surface. The specimen is scanned witha very fine beam of electrons.These are scattered as they hit high and low points in the specimen. The scattered electrons are measured by a detector and used to control a second beam which forms an image on a TV screenElectron sourceT.V. MonitoreElectron beameeeeeeeeSpecimenDetectorVacuum
17 Sample preparation for SEM The preservation used will usually determine whichdrying process to use.There are two basic methods of drying the specimen:Freeze-drying – used after freezingCritical point drying – used after chemical fixation and dehydration
18 Freeze drying for SEMSample placed in nitrogen slush to maintain it’s structureThen placed in liquid nitrogen to allow easy handlingSample placed in copper holder and……….…..placed in freeze drierSample is mounted on a stub
19 Critical point drying for SEM Sample is chemically fixedThen dehydrated with alcoholSample is placed in critical point drier. Here the sample is flushed several times with liquid CO2. The pressure andtemperature is then raised which converts the liquid CO2 to gas. The gas is then vented off slowly.The sample is removed and mounted on a stub
20 Coating the specimen for SEM Most biological specimens are poor conductors and pooremitters of secondary electrons therefore the surface of thesample needs to be coated with a thin layer of a conducting material.There are two ways to do this:Sputter coatingEvaporation of carbon
21 Sputter coating for SEM When power passes to the anode, the noble metal evaporates (called the plasma effect) and the metal falls onto and coats the specimen.ANODEMETALA sputter coaterCATHODE
22 Evaporation of carbon for SEM Two carbon rods are placed end to end. One of the rods issharpened to a point. These are placed in a vacuum and thespecimen is placed below them. When electricity passesthrough the carbon rods, the carbon tip evaporates and thecarbon falls onto and coats the specimen.CARBON RODSCARBON RODS
23 Advantages Disadvantages SEMProvides great depth of focusMicrographs show a 3D image of specimenSmaller and simpler in comparison to TEMOnly surface features seenResolution attainable is not very high (approx 10nmn)
24 Investigating Micrographs Transmission Electron MicrographsScanning Electron MicrographsCan you spot the differences between the two types of electron microscopy?
25 Transmission Electron Micrographs Virus particlesPlasma membraneEndoplasmic reticulumGolgi membranesMitochondrionSection of mammalian cell
26 Scanning Electron Micrographs Sample of geranium petal showing the cone shaped projections and the internal structure.
27 Measuring Micrographs How to work out the size of an organelle?Measured sizeMagnificationMagnification of micrograph is X100000Measured size = 80mmConvert to m = 80000 m100000= 0.8m or 800nm
28 for using this programme. We hope that it has been useful! This programme was developed as part of a work placement project by Sumerah Khan and Sheerin DarianiTHANKS TO:Chris Gilpin, Ian MillerLes Lockey, Samantha Newby
29 ReferencesB. Schotanus (1980) Electron microscopy, what is it ? Marketing electron optics. Philips Export B.V. Eindhoven.Dr Yvonne Miller (1998) Preparation of specimens for TEM and SEM.Mike Mahon, Chris Gilipin, Ian Miller (2000) Microscopy and analysis University of Manchester - School of Biological Sciences.Sam Newby (2000) Freeze drying and critical point drying EMPGU.5) Specimen preparation (1991) (21/1/00) world/SEM/ specimenprep.html pages 1-2.6) Dr. Ron Butler (1980) Transmission electron microscopy, What an SEM is ?, Aims of specimen preparation and Electron microscopy unit. EMPGU
30 1. What is the resolving power of the naked eye? Questions 1 & 21. What is the resolving power of the naked eye?A 200 mB 0.02mC 0.002mD 2mm2. The filament of a transmission electron microscope is made up of which element?A copperB tungstenC carbonD gold
31 True or false? true or false ? Questions 3 & 43. Electrons flow away from the filament because of the large voltage difference between the filament and the anode plateTrue or false?4. In electron microscopy, the lenses used to magnify the image are made of electromagnetstrue or false ?
32 6. A vacuum is needed in the electron microscope to Questions 5 & 65. Which of the following is the first step in the processing of biological material for transmission electron microscopy?A DehydrationB SectioningC FixationD Embedding6. A vacuum is needed in the electron microscope toA Pull the electrons onto the specimenB Eliminate molecules of nitrogen, oxygen or carbon dioxideC Pull the specimen into the columnD Prevent secondary radiation affecting the microscope control panel
33 Questions 7 & 87. The difference between TEM and SEM is that in TEM, secondary low energy electrons are used to produce an image.True or false8. Which of the following structures could not be seen with a light microscope but could be seen with a transmission electron microscope?A NucleusB Cell wallC RibosomeD Golgi apparatus
34 9. Which of the following is not an advantage of TEM? Questions 9 & 109. Which of the following is not an advantage of TEM?A High resolutionB High magnificationC Three dimensional detailD Detail of sub cellular components10. Which of the following statements about SEM is not true?A The specimen is usually coated with goldB Resolution is excellentC Samples for SEM can be chemically fixed or freeze driedD Thin sections of tissue are not necessary
35 wrong please choose again Try againwrong please choose again
36 Answer question 1Answer: A this is the same as the value quoted in the text i.e. 0.2mm
37 Answer question 2Answer: B a tungsten filament is used
39 Answer question 7Answer: this is false as secondary electrons are used to form an image in SEM
40 Answer question 5Answer: the material has to be fixed to prevent distortion and decomposition
41 Answer: B molecules in the air would cause scattering of electrons Answer question 6Answer: B molecules in the air would cause scattering of electrons
42 Answer question 8Answer: C it is possible to see the other structures with a light microscope
43 Answer question 9Answer: C TEM gives a two dimensional image
44 Answer: B SEM has relatively poor resolution compared with TEM Answer question 10Answer: B SEM has relatively poor resolution compared with TEM
45 Scanning Electron Micrographs Sample of geranium petal showing the cone shaped projections and the internal structure.
46 Transmission Electron Micrographs Virus particlesPlasma membraneEndoplasmic reticulumGolgi membranesMitochondrionSection of mammalian cell
47 Q 11What is the size of the virus Questions 11 & 12Q 11What is the size of the virusQ12 What is the size of the mitochondrionMagnification X100000Magnification X13000Measured size = 9mmMeasured size = 11mmA 90mmA 846mmB 90nmB 8.46nmC 90mC 8.46mD 900nmD 846nm