Molecular Biology - Appendix - Microscopy

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microscopy

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the use of the microscope for investigation

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Robert Hooke

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observed the first cells through a microscope

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wavelength

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the distance from the crest of one wave the crest of the next wave

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interference

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the process by which two or more waves combine to reinforce or cancel one another producing a weight equal to the sum of the two combining waves

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diffraction

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additive or canceling interference of light waves

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focal length

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the distance between the midline of the lens and the point at which rays passing through the lens converge to a focus

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diopter

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lens magnifying strength

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Dr.

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the inverse of the focal length, measured in meters

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angular aperture

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half-angle alpha of the cone of light entering the objective lens of the microscope from the specimen

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resolution

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the minimum distance that can separate two points that still remain identifiable as separate points when viewed through the microscope

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abbe equation

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.61 lambda /n sin alpha

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abbe equation

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formula for relating wavelength, resolution, refractive index and angular aperture

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limit of resolution

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best possible resolution. Can be increased with oil immersion lens

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compound microscope

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common laboratory microscope

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condenser lenses

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direct light toward a specimen mounted on a glass slide and positioned on the stage of the microscope

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ocular lens

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eyepiece

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intermediate lens

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positioned between the objective and ocular lenses to accomplish further enlargement

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brightfield microscopy

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common form of light microscopy

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phase contrast microscopy

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improves contrast without sectioning and staining exploiting differences in the thickness and refractive index of various regions in the cells being examined

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differential interference contrast microscopy

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employs a special prism split the illuminating light beam into two separate rays. The image appear three dimensional due to a shadow casting illusion

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fluorescence

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process that begins with the absorption of light by molecule and ends with the emission of light with a longer wavelength

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fluorescence microscopy

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employs light to excite fluorescence in the specimen using excitation filter between the light source condenser lens transmits only light of a particular wavelength

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immunostaining

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technique based on the ability of antibodies to recognize and bind to specific molecules. Molecules of antibodies are called antigens. Antibodies are proteins produced naturally by the immune system in response to invading microorganisms or foreign molecules but they can also be generated in the laboratory by injecting a foreign protein

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immunostaining

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common application of fluorescent probes

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indirect immunofluorescence

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a tissue or cells treated with antibody is not labeled with the eye. This antibody, called primary antibody, attaches to specific antigenic sites within the tissue or cell. The second type of antibody, called secondary antibody, is then added. The secondary antibodies labeled with fluorescent dye and attaches to the primary antibody. Because more than one primary antibody molecules attached to it antigen, and more than one secondary antibody molecules and attached each primary antibody, more fluorescent molecules are concentrated near each molecule that we seek to detect.

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confocal microscope

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employs a laser beam to produce an image of a single plane of the specimen at a time. This improves the resolution along the optical axis of the microscope

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digital microscopy

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microscopic images are recorded and stored electronically by placing a video or digital camera and image plane produced by the ocular lens. The resulting images can be enhanced by a computer. Specimen does not need to be killed by fixation

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microtome

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used to slice specimen into thin sections a few micrometers thick

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transmission electron microscope

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forms an image from electrons that are transmitted through the specimen

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TEM

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transmission electron microscope

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scanning electron microscope

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generates an image by scanning specimen surface with a beam of electrons

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SEM

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scanning electron microscope

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high-voltage electron microscope

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similar to a transmission electron microscope that uses an accelerating voltage that is much higher. Thicker specimens can be examined with good resolution.

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ultramicrotome

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used to the slice ultrathin sections of a specimen suitable for electron microscopy

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immuno electron microscopy

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the linking of antibodies to heavy metals, e.g. colloidal gold, to make them visible to electron microscopy

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Nucleic acid hybridization allows a researcher to . . .

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identify and isolate specific DNA or RNA molecules

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Bond energy is . . .

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. . . expressed in moles