Microscopy

• Microscopy: understand the difference between m, cm, mm, μm and nm.
• Microscopy: describe the difference between compound and stereo light microscopes.
• Microscopy: describe some techniques used to enhance image quality.
• Transmission Electron Microscope (TEM) produces detailed two-dimensional images of the cell interior.
• An electron beam passes through a thin section of the specimen in a Transmission Electron Microscope (TEM).
• Parts of the specimen absorb electrons and appear dark in a Transmission Electron Microscope (TEM).
• Other parts of the cell allow electrons to pass through in a Transmission Electron Microscope (TEM).
• Scanning Electron Microscope (SEM) produces three-dimensional images of the surface of cells and organisms.
• In a Scanning Electron Microscope (SEM), an electron beam is directed onto the surface of the specimen from above.
• The specimen is dried and coated with a metal to produce secondary electrons in a Scanning Electron Microscope (SEM).
• An electron beam scans the specimen surface and electrons are scattered in a Scanning Electron Microscope (SEM).
• Scanning Electron Microscope (SEM) creates incredible images.
• Tardigrade (water bear) is an example of an organism imaged by a Scanning Electron Microscope (SEM).
• Light Microscopy: understand the difference between m, cm, mm, μm and nm.
• Light Microscopy: describe the difference between compound and stereo light microscopes.
• Light Microscopy: describe some techniques used to enhance image quality.
• Electron beam is produced by a hot wire in the cathode (electron gun).
• Electrons are attracted to positive anodes to create an electron beam.
• Objective and condenser lenses are electromagnets that focus the electron beam onto the specimen.
• Projector lens directs electron beam to the viewing screen.
• Specimens are killed and fixed using glutaraldehyde and osmium tetroxide to create covalent crosslinks and stabilise lipid bilayers.
• Electrons have poor penetrating power therefore thin sections are created using monomeric resin and microtomes.
• Image clarity is dependent on having a range of electron densities within the cell.
• Electron densities are exaggerated by impregnating cells with heavy metal salts, such as uranium and lead.
• Fixation, sectioning, rehydration, and mounting are not the normal process of slide preparation.
• Water does not scatter light which causes photon noise.
• Water has multiple densities therefore light is not scattered.
• Water scatters light and causes photon noise.
• Water has a single density therefore light is not scattered.
• Heavy metal salts are added to specimens before they are viewed under an electron microscope to make the specimen heavy and prevent it from floating away in the vacuum.
• Heavy metals create differences in electron density and image contrast.
• A vacuum must be maintained in both Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM) as electrons will deflect off air particles.
• Stereo light microscope is used for visualising larger specimens and designed for viewing surface features with a long 'working distance' that limits magnification and only one objective lens.
• Compound light microscope has a short working distance that means greater magnification and can have multiple objective lenses.
• Compound light microscopy is used to visualise items not visible to the naked eye and light is directed from under the specimen.
• Specimens can be living or dead but often need to be stained to increase contrast.
• Microscopy using light microscopes involves ensuring the surface is flat, putting the lowest power objective lens in place, adjusting the light source to 2/3 maximum, opening the diaphragm fully to maximise light, placing the slide on the stage and positioning it to illuminate.
• Refraction changes the direction that light travels in and occurs when the density of the object changes.
• Oil immersion lens is used to reduce refraction when light moves through multiple media of differing densities/refractive indices and causes light to 'miss' the objective lens, limiting resolution at high magnification.
• Fixation in microscopy preserves specimens in a lifelike state using chemicals or heat.