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E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of.

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Presentation on theme: "E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of."— Presentation transcript:

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2 E LECTRON M ICROSCOPY Advantages and disadvantages of TEM Advantages and disadvantages of SEM Introduction Principles of operation of TEM Principles of operation of SEM Sample preparation for TEM Sample preparation for SEM Investigating micrographs School of Biological Sciences

3 INTRODUCTION With the invention of the light microscope it was discovered that plant and animal tissues were made up of aggregates of individual cells. However, light microscopes are limited to resolution. approximately x1000 magnification and have poor resolution. Therefore not all the internal structures of a cell can be seen with 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.

4 RESOLUTION When there is sufficient light, two points 0.2mm apart or more can be distinguished with the naked eye as being separate points. When this distance is less than 0.2mm, only one point is seen. This distance is called the resolving power (or resolution) of the eye. In other words resolution is the closeness two objects can be in proximity and still be perceived as two separate objects. Can’t see two separate objects Can see two separate objects

5 The invention of the electron gun led to the development of the electron microscope. The metal tungsten filament is heated to about 2500 o C which causes it to release electrons. Due to the large voltage difference between the filament and the anode plate the electrons are forced to flow in the direction of the arrow. The cathode shield increases the electron flow further and concentrates the electrons into a narrow beam. Anode Electron beam Cathode Filament High voltage generator Back to principles of operation

6 TEM TransmissionSEM Scanning TEM 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.internal structures Two main types of electron microscopes; 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.three dimensional image

7 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.

8 PRINCIPLES OF OPERATION OF TEM Specimen holder Electron gun Projection chamber

9 THE ELECTRON GUN – produces an electron beam. THE COLUMN – uses electromagnetic lenses to control the beam and produce a magnified image IMAGE VIEWING AND RECORDING The image is produced on a fluorescent screen below which a shutter and camera are located. Condenser lens The main components of a TEM are: Objective lens Projector lens Specimen Fluorescent screen Vacuum e e e e ee e e e e e e e e e e e

10 Electrons only behave like light when they are manipulated in vacuum. Therefore the whole column is evacuated since atoms such as O 2 and CO 2 scatter the electrons. ee ee e e e

11 SAMPLE PREPARATION FOR TEM The aims of sample preparation are as follows: To preserve the material in its natural state To ensure that the material withstands changes which might occur on exposure to atmosphere, vacuum and electron beam.

12 Fixation for TEM The tissue is cut into tiny pieces It is then placed into fixing solution

13 Dehydration and embedding of TEM Tissue is dehydrated in alcohol It is then placed in a dilute solution of resin embedding media Specimen vials Tissue is placed in final embedding mixture and the resin is polymerised in the oven

14 Section cutting of TEM Sections 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

15 Advantages Disadvantages Thin sections are effectively two dimensional slices of tissue and do not convey the three dimensional arrangement of cellular components Very good resolution Can see sub-cellular components and measure them magnification Artefacts may be created TEM

16 PRINCIPLES OF OPERATION OF SEM Control panel Image viewing Specimen chamber Electron gun Detector

17 SEM uses electrons that are emitted from the specimen surface. The specimen is scanned with a 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 screen Specimen Vacuum Detector e e e e e e eee Electron source Electron beam T.V. Monitor

18 Sample preparation for S EM The preservation used will usually determine which drying process to use. There are two basic methods of drying the specimen: Critical point drying – used after chemical fixation and dehydration Freeze-drying – used after freezing

19 Freeze drying for S EM Then placed in liquid nitrogen to allow easy handling Sample placed in nitrogen slush to maintain it’s structure Sample is mounted on a stub Sample placed in copper holder and………. …..placed in freeze drier

20 Critical point drying for SEM Sample is chemically fixed Then dehydrated with alcohol Sample is placed in critical point drier. Here the sample is flushed several times with liquid CO 2. The pressure and temperature is then raised which converts the liquid CO 2 to gas. The gas is then vented off slowly. The sample is removed and mounted on a stub

21 Coating the specimen for SEM Most biological specimens are poor conductors and poor emitters of secondary electrons therefore the surface of the sample needs to be coated with a thin layer of a conducting material. There are two ways to do this: Sputter coating Evaporation of carbon

22 Sputter coating 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.METAL ANODE CATHODE A sputter coater

23 Evaporation of carbon for SEM Two carbon rods are placed end to end. One of the rods is sharpened to a point. These are placed in a vacuum and the specimen is placed below them. When electricity passes through the carbon rods, the carbon tip evaporates and the carbon falls onto and coats the specimen. CARBON RODS

24 Advantages Disadvantages Provides great depth of focus Micrographs show a 3D image of specimen Smaller and simpler in comparison to TEM Only surface features seen Resolution attainable is not very high (approx 10nmn) SEM

25 Investigating Micrographs Transmission Electron Micrographs Scanning Electron Micrographs Can you spot the differences between the two types of electron microscopy?

26 Transmission Electron Micrographs Mitochondrion Plasma membrane Endoplasmic reticulum Golgi membranes Virus particles Section of mammalian cell

27 Scanning Electron Micrographs Sample of geranium petal showing the cone shaped projections and the internal structure.

28 Measuring Micrographs Magnification of micrograph is X How to work out the size of an organelle? Measured size Magnification   m = 0.8  m or 800nm Measured size = 80mm Convert to  m = 80000

29 for using this programme. We hope that it has been useful! THANKS TO: Chris Gilpin, Ian Miller Les Lockey, Samantha Newby This programme was developed as part of a work placement project by Sumerah Khan and Sheerin Dariani

30 References 1)B. Schotanus (1980) Electron microscopy, what is it ? Marketing electron optics. Philips Export B.V. Eindhoven. 2)Dr Yvonne Miller (1998) Preparation of specimens for TEM and SEM. 3)Mike Mahon, Chris Gilipin, Ian Miller (2000) Microscopy and analysis University of Manchester - School of Biological Sciences. 4)Sam Newby (2000) Freeze drying and critical point drying EMPGU. 5) Specimen preparation (1991) (21/1/00) universe/Academic world/SEM/ specimenprep.html pages 1-2.http://www.lifelong.com/lifelong- universe/Academic world/SEM/ specimenprep.html 6) Dr. Ron Butler (1980) Transmission electron microscopy, What an SEM is ?, Aims of specimen preparation and Electron microscopy unit. EMPGU

31 Questions 1 & 2 1. What is the resolving power of the naked eye? A200  m B0.02m C0.002m D2mm 2. The filament of a transmission electron microscope is made up of which element? Acopper Btungsten Ccarbon Dgold

32 Questions 3 & 4 3. Electrons flow away from the filament because of the large voltage difference between the filament and the anode plate Trueor false? 4. In electron microscopy, the lenses used to magnify the image are made of electromagnets true or false ?

33 Questions 5 & 6 5. Which of the following is the first step in the processing of biological material for transmission electron microscopy? ADehydration BSectioning CFixation DEmbedding 6. A vacuum is needed in the electron microscope to APull the electrons onto the specimen BEliminate molecules of nitrogen, oxygen or carbon dioxide CPull the specimen into the column DPrevent secondary radiation affecting the microscope control panel

34 Questions 7 & 8 7. The difference between TEM and SEM is that in TEM, secondary low energy electrons are used to produce an image. True or false 8. Which of the following structures could not be seen with a light microscope but could be seen with a transmission electron microscope? ANucleus BCell wall CRibosome DGolgi apparatus

35 Questions 9 & Which of the following is not an advantage of TEM? AHigh resolution BHigh magnification CThree dimensional detail DDetail of sub cellular components 10. Which of the following statements about SEM is not true? A The specimen is usually coated with gold B Resolution is excellent C Samples for SEM can be chemically fixed or freeze dried D Thin sections of tissue are not necessary

36  Try again wrong please choose again

37  Answer question 1 Answer: A this is the same as the value quoted in the text i.e. 0.2mm

38  Answer question 2 Answer: B a tungsten filament is used

39  Answer correct Correct well done

40  Answer question 7 Answer: this is false as secondary electrons are used to form an image in SEM

41  Answer question 5 Answer: the material has to be fixed to prevent distortion and decomposition

42  Answer question 6 Answer: B molecules in the air would cause scattering of electrons

43  Answer question 8 Answer: C it is possible to see the other structures with a light microscope

44  Answer question 9 Answer: C TEM gives a two dimensional image

45  Answer question 10 Answer: B SEM has relatively poor resolution compared with TEM

46 Scanning Electron Micrographs Sample of geranium petal showing the cone shaped projections and the internal structure.

47 Transmission Electron Micrographs Mitochondrion Plasma membrane Endoplasmic reticulum Golgi membranes Virus particles Section of mammalian cell

48 Magnification X13000 Magnification X Q12 What is the size of the mitochondrion Q 11What is the size of the virus Measured size = 9mm Measured size = 11mm Questions 11 & 12 D900nm D900nm C90  m C90  m A90mm A90mm B90nm B90nm A846mm A846mm B8.46nm B8.46nm C8.46  m C8.46  m D846nm D846nm


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