S200 Course LECTURE 1 TEM

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S200 Course LECTURE 1 TEM

Development of Electron Microscopy 1897 Discovery of the electron (J.J. Thompson) 1924 Particle and wave theory (L. de Broglie) 1926 Electromagnetic Lens (H. Busch) 1932 Construction of first T.E.M. (E. Ruska) 1938 First S.E.M. constructed (M. von Ardenna) 1939 First commercial T.E. M. 1960 s (early) First Commercial S.E.M. Resolution a little better than that of L.M. Resolution 24 Å Resolution of present research T.E.M s < 2 Å S.E.M s < 5 Ă

DIMENSIONS OF CERTAIN BIOLOGICAL STRUCTURES AS SEEN BY ELECTRON MICROSCOPY Mammalian Oocyte 100µm (0.1mm) Liver Cell 20µm Red Blood Cell 7µm Mitochondria/Some Bacteria 0.2-0.5µm (2-500nm) Ribosomes/Certain Viruses 20nm Cell Membrane (Width) 10nm DNA Molecule 2nm (20Å)

MICROSCOPY RESOLVING POWER (RESOLUTION) Smallest distance by which two points on a specimen can be separated and still be distinguished as two points.

LIGHT MICROSCOPY RESOLUTION Maximum resolving power of a microscope = half wavelength of illuminating beam. Wavelengths of visible light 0.4-0.7µm In electron microscope wavelength dependent on accelerating voltage of electrons (40-100Kv) Theoretical resolution 0.002nm Actual resolution approx. 0.2nm (2Å)

Hitachi H-7000 Scanning Transmission Electron Microscope

BASIC ELECTRON MICROSCOPE Evacuated metal cylinder within which are aligned, one under another: 1. Tungsten filament (the cathode) 2. A Metal plate with central aperture (the anode) 3. A number of magnetic lenses 4. A Fluorescent screen 5. A photographic plate

Transmission Electron Microscope

THE TRANSMISSION ELECTRON MICROSCOPE Electron Gun 1. Tungsten filament V (Cathode) 2. Metal housing (cathode shield Wehnelt cylinder) aperture approximately 1mm diameter. 3. Anode plate circular plate with aligned aperture.

The Electron Gun

THE TRANSMISSION ELECTRON MICROSCOPE Choice of accelerating Voltage (AV) Higher AV:- Better penetration of specimen Increased electron gun brightness Improved phosphor screen efficiency Energy loss in specimen decreases loss in resolution due specimen radiation damage Lower specimen contrast Less high voltage stability

ELECTRON MICROSCOPY Accelerating Voltage TEM 60kV 100kV (SEM 5kV 20kV)

Accelerating Voltage Effect on specimen contrast (A) 80kV (B) 40kV

Magnetic Lenses

THE TRANSMISSION ELECTRON MICROSCOPE Magnetic Lenses (1) 1. Coil of several thousand turns of wire through which a current of less than or equal to one amp is passed --- creates a magnetic field. 2. Electrons are deflected by magnetic field. 3. To avoid need for a large current passing through the coil the magnetic field is concentrated by encasing the coil in a soft iron cover with only a small ring-shaped gap in centre. The entire field is therefore concentrated in this gap.

THE TRANSMISSION ELECTRON MICROSCOPE Magnetic Lenses (2) 4. To concentrate field further a soft iron pole piece is inserted into the bore of the objective lens reducing the bore of the lens and width of the ring-shaped gap. 5. To focus an electron beam onto a given plane the current through the coils must be set to a precise value. current beam focus closer to lens current beam focus further from lens

THE TRANSMISSION ELECTRON MICROSCOPE Condensor Lens Magnetic Lens (3) To illuminate the specimen. Relatively weak lens. Longer focal length than objective or projector lens. May bring electron beam into focus directly upon specimen, above the specimen (over focusing) or below the specimen (under focusing).

Specimen THE TRANSMISSION ELECTRON MICROSCOPE OBJECTIVE LENS Lenses Strong lens. Highly concentrated magnetic field and short focal length. Causes electron beam, which has passed through specimen, to focus at a point a few mm below specimen. Magnification of image produced a short distance below focused point.

THE TRANSMISSION ELECTRON MICROSCOPE Lenses Projector Lens Magnification produced by projector lens dependent on current passing through the coil of the lens (ie increase current spreads beam further = higher mag.) Projector lens has great depth of focus (several meters). Therefore distance at which fluorescent screen or photographic plate are from lens is not critical.

THE TRANSMISSION ELECTRON MICROSCOPY Fluorescent Screen Fluorescence property of emitting radiation under the influence of electromagnetic or electron beam bombardment. In the TEM screen coated with a material in the visible range, eg zinc sulphide, is installed beneath the projector lens in the path of the electron beam. Screen emits visible light when bombarded with electrons. Deflected electrons eliminated from beam remain dark (deflected by specimen components)

THE TRANSMISSON ELECTRON MICROSCOPE Fluorescent Screen The resolution of the fluorescent screen is limited to 70-100µm by the grain size of the fluorescent material and by light scattering within this material. Therefore: PHOTOGRAPHIC PLATE specimens are photographed with fine-grain emulsion ensures recording of all detail inherent in the electron image.

THE TRANSMISSION ELECTRON MICROSCOPE Vacuum System Electrons can travel only a few µm in air before they are stopped or slowed down by collisions with gas molecules. Distance between electron gun and photographic is approximately 1 metre. Electron Microscope must be evacuated (10-4 Torr) Two types of vacuum pump used:- 1. Rotary (mechanical) fore-pump. 2. Diffusion pump (oil or mercury).

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