Chapter 18

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Biological Sciences
David Harrison

Cell Biology – Chapter 18 18.1 – The Light Microscope A light microscope consists of a light source, which may be external to the microscope or build in the base, that illuminates the specimen. The substage condenser lens gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light. Light rays focused on specimen in condenser lens are collected by objective lens. The image formed by the object lens is used as on object by a second lens, called the ocular lens which enlarges image. Third lens is located in front part of the eye and uses the virtual image to produce a real image in the retina. The total magnification attained by the microscope is the product of the magnifications produced by the objective lens and the ocular lens. Resolution To see an image in the microscope and to count how many there are for say chromosomes, you would have to use a less magnification because you want to spread the image over a greater part of your retina. The more photoreceptors that provide information about the image, the more detail that can be seen . The change of ocular fails to provide additional information because the image produced by the objective lens does not possess any further detail to be enhaced by the increased power of the ocular lens. Second switch provides empty magnification. Opitcal quality is measured by the exten to which the fine detail present in a specimen can be discriminated, or resolved. Resolution attained is limited by diffraction. Thus the resolving power of a microscope can be defined in terms of the ability to see two neighbouring points in the visual field as two distinct entities. . Resolving power is alos affected by optical flaws, or aberrations. Visibility Contrast or the difference in appearance between adjacent parts of an object or between an object and its background. Best ways is to make a thin, translucent specimen visible under the microscope is to stain it with a dye. A problem with stains is that they generally cannot be used with living cells, they are toxic, or the stains do not penetrate the plasma membrane. Bright field microscope, the cone of light that illuminates the specimen is seen as a bright background against which the image of the specimen must be contrasted. They are ideally suited for specimens of high contrast, but it may not provide optimal visibility for other specimens. Preparation of Specimens for Bright Field Light Microscope Specimens to be observed are divided in two categories: whole mount is an intact object, either living or dead, and can consist of an entire microscopic organism or a small part of a larger organism. Most tissues need to be examined as a very thin slice or section. Cells must first immerse in a chemical solution (fixative). A good fixative rapidly penetrates the cell membrane and immobilizes all of its macromolecular material. After fixation it is dehydrated by transfer through a series of alcohol and paraffin (wax). Phase- Contrast Microscopy Small, unstained specimens can be difficult to see. Phase contrast microscope makes highly transparent objects more visible. Can distinguish different parts of an object because they affect light differently. Phase contrast microscope converts differences in refractive index into differences in intensity. They 1. Separate the direct light that enters the objective lens form the diffracted light emanating from the specimen and causes the light rays from these two sources to interfere with one another. Is used everyday in research. A phase microscope is a type of interference microscope that has optical handicaps that result in loss of resolution. Other interference microscope minimize optical artifacts. Differential interference contrast (DIC) delivers an image that has an apparent three dimensional quality. Fluorescence Microscopy (and Related Fluorescence – Based Techniques) Light microscope has been transformed from an instrument designed primarily to exam sections of fixed tissues to one capable of observing the dynamic events occurring at the molecular level in living cells. The fluorescence microscope allows viewers to observe the location of certain compounds. Fluorochromes absorb invisible, ultraviolet radiation and release a portion of the energy in the longer, visible wavelengths, phenomenon called fluorescence. Objects stained with a fluorochrome appear brightly coloured against a black
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