Chapter 18 - Techniques in Cell and Molecular Biology.docx

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Biological Sciences
Rene Harrison

Techniques in Cell and Molecular Biology o Microscopes are instruments that produce enlarged image of an object.  A light source, which illuminates the specimen, is critical within the microscope.  Condenser lens gathers the diffuse ray from the light source and illuminates the specimen with a small cone of bright light that allows very small parts of the specimen to be seen after magnification  Light rays focused on the specimen by the condenser lens are then collected by the microscope’s objective lens.  Two sets of light rays that enter objective lens: those that the specimen had altered and those that it hasn’t  Image formed by the objective lens is used as an object by a second lens system, the ocular lens, to form an enlarged and virtual image  Total magnification attained by the microscope is the product of the magnifications produced by the objective lens and the ocular lens o Relatively high-power objective lens and an ocular lens that magnifies the image of the objective lens another fivefold – change in oculars to increase the size of the object being view. If you were to switch from the 5X to 10X ocular, you would most likely increase your ability to determine the number of details present within cell.  Details produced by the objective lens over a greater part of your retina  The more photoreceptors that provide information about the image, the more detail that can be seen  Second switch in oculars provides only empty magnification  Optical quality of an objective lens is measured by the extent to which the fine detail present in a specimen can be discriminated, or resolved  Resolution attained by a microscope is limited by diffraction. Since the diffraction of light emanating from a point in the specimen can never be seen as a point in the image, but only as a small disk. If the disks produced by two nearby points overlap, the points cannot be distinguished in the image o More practical side of microscopy from that of limits of resolution is the subject of visibility, which is concerned with factors that allow an object actually to be observed  We see through a window or through a microscope are those objects that affect the light differently from their background  Contrast, or different in appearance between adjacent parts of an object or between an object and its background.  Through microscopes, we examine objects by having the light fall on them and then observing the light  We place the specimen between the light source and our eyes and view the light that is transmitted through the object  One of the best ways to make thin, translucent specimen visible under the microscope is to stain it with a dye, which absorbs only certain wavelengths within the visible spectrum  Wavelengths that are not absorbed are transmitted to the eye, causing the stained object to appear clotted  Feulgen stain, which is specific for DNA, causing the chromosomes to appear colored under the microscope  A problem with stains is that they generally cannot be used with living cells; they are usually toxic, or the staining conditions are toxic, or the stains do not penetrate the plasma membrane  Different types of light microscopes use different types of illumination.  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  Ideally suited for specimens of high contrast, such as stained sections of tissues, but it may not provide optimal visibility for other specimens o Specimens to be observed with the light microscope are broadly divided into two categories: a. Whole mount—is an intact object, either living or dead, and can consist of an entire microscopic organism such as a protozoan or a small part of a larger organism b. Sections – most plants and animal cells are much too opaque for microscopic analysis unless examined as a very thin slice  In order to prepare a section, the cells are first killed by immersing the tissue in a chemical solution, called a fixative which rapidly penetrates the cell membrane and immobilizes all of its macromolecular material so that the structure of cell is maintain as close as possible to that of its live state.  Common fixatives include: formaldehyde, alcohol, or acetic acid  Tissue is dehydrated through a series of alcohols and then usually embedded in paraffin, which provides mechanical support during sectioning  After the paraffin sections are immersed in toluene, which dissolves the wax, leaving the thin slice of tissue attached to the slide, where it can be stained or treated with antibodies or other agents o Phase-contrast microscope makes highly transparent objects more visible  Can distinguish different parts of an object because they affect light differently from one another  One basis for difference is the refractive index; different cells hold different proportions of DNA, RNA, protein, lipid, carbohydrate, salts, and water. Regions of different composition are likely to have different refractive indexes  Phase-contrast microscope converts differences in refractive index into differences in intensity, which are visible to the eye.  Phase-contrast microscope accomplishes this in two ways: a. Separating the direct light that enters the objective lens from the diffracted light emanating from the specimen b. Causing light rays from these two sources to interfere with one another  Ideally it reflects the way in which the light from that part of specimen interferes with the direct light  Most useful when examining intracellular components of living cells at relatively high resolution  Optical handicaps that result in loss of resolution, and image suffers from interfering halos and shading produced where sharp changes in refractive index occur o The phase-contrast microscope is a type of interference microscope. Other types of interference microscopes minimize these optical artifacts by achieving a complete separating of direct and diffracted beams using complex light paths and prisms.  Differential interference contrast (DIC), or sometimes Nomarski interference after its developer, delivers an image that has an apparent 3-D quality.  Depends on the rate of change of refractive index across a specimen.  Consequence, the edges of structures, where the
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