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10.2 electron microscope Principles and limitations of scanning and transmission EM Difference between magnification and resolution

Magnification How much larger the image is compared to the original object How much larger the image is compared to the original object Magnification = size of image size of object

Resolution and the electron microscope Instead of light the electron microscope uses a beam of electrons Instead of light the electron microscope uses a beam of electrons Electrons have a much shorter wavelength than light Electrons have a much shorter wavelength than light They can pass between objects that are too close together to let light pass between them They can pass between objects that are too close together to let light pass between them The resolution in a light microscope is limited to 2µm, but a transmission electron microscope can resolve 1nm The resolution in a light microscope is limited to 2µm, but a transmission electron microscope can resolve 1nm

Electron microscope video 1. Why is electron microscope important? 2. Name the photo from an electron microscope. 3. On the screen what is a dark patch representitive of in the specimen? 4. What do scientists add to ‘stain’ the specimen? 5. Why is there a vacuum in the EM? 6. Why are the specimens mounted on a grid? 7. Why is the specimen embedded in resin?

Transmission electron microscope TEM Object has to be really thin Object has to be really thin Stained with electron dense stain (e.g. heavy metals –lead citrate and uranyl acetate) Stained with electron dense stain (e.g. heavy metals –lead citrate and uranyl acetate) The electrons pass down the microscope, are focused with electro magnets, and either pass through the specimen (shines bright on the screen below) or are absorbed by the stain (appears dark on the screen) The electrons pass down the microscope, are focused with electro magnets, and either pass through the specimen (shines bright on the screen below) or are absorbed by the stain (appears dark on the screen) There is a vacuum in the microscope to prevent the electrons from being deflected There is a vacuum in the microscope to prevent the electrons from being deflected

ANIMAL CELL PLANT CELL Courtesy of Dr. Julian Thorpe – EM & FACS Lab, Biological Sciences University Of Sussex Transmission electron micrograph

LIGHT MICROSCOPE TRANSMISSION ELECTRON MICROSCOPE SCANNING ELECTRON MICROSCOPE Image on viewing screen Image on fluorescent screen Image viewed directly light source glass condenser lens specimen objective lens detector cathode gun (source of electrons) specimen eyepiece lens electromagnetic condenser lens projector lens beam deflector

LIGHT MICROSCOPE TRANSMISSION ELECTRON MICROSCOPE SCANNING ELECTRON MICROSCOPE Image on viewing screen Image on fluorescent screen Image viewed directly light source glass condenser lens specimen objective lens detector cathode gun (source of electrons) specimen eyepiece lens electromagnetic condenser lens projector lens beam deflector

The SEM is designed for the study of the surfaces of solid objects such as insects and pollen grains In contrast to the TEM that transmits electrons through the specimen, the electron beam in the SEM is scanned across the specimen surface The electrons do not pass through the specimen because of its thickness and because heavy metal (for example, gold) is evaporated onto the surface The electron beam is reflected as it strikes the specimen Special detectors collect scattered electrons and create electrical signals that are displayed on a computer monitor A three-dimensional image of the specimen’s surface is obtained Scanning electron microscope SEM Link to virtual SEM

Comparing different types of microscopes FeatureOpticalTEMSEM Level of Resolution Colour/ B&W Hazard level to user (include ref to type of stains used) Living or dead specimen? 2D or 3D image? Portability? Likelihood of introduction of atrefacts

OpticalTEMSEM Lower resolving power Best resolving power – most detail seen 2 nd best resolving power colour Black and white (any colour is added with a computer) Less hazardous Stains can be very poisonous (accumulative) + other hazards Living cells and movement can be observed Dehydration because of vacuum means dead material only 3d or 2d possible Only 2d Only 3d Portable – use in field Not portable Less steps in preparation – less chance of artefacts Most chance of artefacts (distorting original specimen) Dehydration will cause some artefacts Comparing different types of microscopes

Courtesy of Leanne Cooper The University of Lancaster The three dimensional structure of a red and white blood cell can clearly be seen on the surface of the tissue Scanning electron micrograph

SEM and TEM images are black and white. Micrographs of these images are often artificially coloured to highlight certain structures and enhance the micrograph False colour micrograph of an SEM image