Viruses are acellular bioactive parasites that attack virtually every form of cellular life. Their shape is either pseudospherical with icosahedral symmetry, as in the poliomyelitis virus, or rodlike, as in the tobacco mosaic virus TMV. A virus surrounded only by protein coat capsid is a naked virus; some viruses e.
Figure Multiple-fluorescent-probe microscopy. In this composite micrograph of a cell in mitosis, three different fluorescent probes have been used to stain three different cellular components. The spindle microtubules are revealed with a green fluorescent antibody, more Important methods, discussed later in the chapter, enable fluorescence microscopy to be used to monitor changes in the concentration and location of specific molecules inside living cells see p.
Antibodies Can Be Used to Detect Specific Molecules Antibodies are proteins produced by the vertebrate immune system as a defense against infection discussed in Chapter They are unique among proteins because they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule or antigen.
The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. When labeled with fluorescent dyes, they are invaluable for locating specific molecules in cells by fluorescence microscopy Figure ; labeled with electron -dense particles such as colloidal gold spheres, they are used for similar purposes in the electron microscope discussed below.
The sensitivity of antibodies as probes for detecting and assaying specific molecules in cells and tissues is frequently enhanced by chemical methods that amplify the signal. For example, although a marker molecule such as a fluorescent dye can be linked directly to an antibody used for specific recognition—the primary antibody—a stronger signal is achieved by using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it Figure Figure Indirect immuno-cytochemistry.
This detection method is very sensitive because the primary antibody is itself recognized by many molecules of the secondary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily more The most sensitive amplification methods use an enzyme as a marker molecule attached to the secondary antibody.
The enzyme alkaline phosphatasefor example, in the presence of appropriate chemicals, produces inorganic phosphate and leads to the local formation of a colored precipitate.
This reveals the location of the secondary antibody that is coupled to the enzyme and hence the location of the antibody- antigen complex to which the secondary antibody is bound. Since each enzyme molecule acts catalytically to generate many thousands of molecules of product, even tiny amounts of antigen can be detected.
An enzyme-linked immunosorbent assay ELISA based on this principle is frequently used in medicine as a sensitive test—for pregnancy or for various types of infections, for example. Although the enzyme amplification makes enzyme-linked methods very sensitive, diffusion of the colored precipitate away from the enzyme means that the spatial resolution of this method for microscopy may be limited, and fluorescent labels are usually used for the most precise optical localization.
Antibodies are made most simply by injecting a sample of the antigen several times into an animal such as a rabbit or a goat and then collecting the antibody-rich serum.
This antiserum contains a heterogeneous mixture of antibodies, each produced by a different antibody-secreting cell a B lymphocyte. The different antibodies recognize various parts of the antigen molecule called an antigenic determinant, or epitopeas well as impurities in the antigen preparation.
The specificity of an antiserum for a particular antigen can sometimes be sharpened by removing the unwanted antibody molecules that bind to other molecules; an antiserum produced against protein X, for example, can be passed through an affinity column of antigens Y and Z to remove any contaminating anti-Y and anti-Z antibodies.
Even so, the heterogeneity of such antisera sometimes limits their usefulness. This problem is largely overcome by the use of monoclonal antibodies see Figure However, monoclonal antibodies can also have problems. Since they are single antibody protein species, they show almost perfect specificity for a single site or epitope on the antigen, but the accessibility of the epitope, and thus the usefulness of the antibody, may depend on the specimen preparation.
For example, some monoclonal antibodies will react only with unfixed antigens, others only after the use of particular fixatives, and still others only with proteins denatured on SDS polyacrylamide gels, and not with the proteins in their native conformation.
Imaging of Complex Three-dimensional Objects Is Possible with the Optical Microscope For ordinary light microscopy, as we have seen, a tissue has to be sliced into thin sections to be examined; the thinner the sectionthe crisper the image.
In the process of sectioning, information about the third dimension is lost. How, then, can one get a picture of the three-dimensional architecture of a cell or tissue, and how can one view the microscopic structure of a specimen that, for one reason or another, cannot first be sliced into sections?
This can make it very hard to interpret the image in detail, and can lead to fine image structure being obscured by the out-of-focus light. Two approaches have been developed to solve this problem: These three-dimensional microscopic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting the light that comes from out-of-focus regions above and below that plane.
Thus one sees a crisp, thin optical section. From a series of such optical sections taken at different depths and stored in a computer, it is easy to reconstruct a three-dimensional image. The methods do for the microscopist what the CT scanner does by different means for the radiologist investigating a human body: The computational approach is often called image deconvolution.
To understand how it works, remember how the wave nature of light means that the microscope lens system gives a small blurred disc as the image of a point light source, with increased blurring if the point source lies above or below the focal plane.
This blurred image of a point source is called the point spread function. An image of a complex object can then be thought of as being built up by replacing each point of the specimen by a corresponding blurred disc, resulting in an image that is blurred overall.
For deconvolution, we first obtain a series of blurred images, focusing the microscope in turn on a series of focal planes—in effect, a blurred three-dimensional image.Tests Test Name Alternative Names Included Tests Medical Necessity Applies PHL Test Code View Details (Example Test) - Test Name (Required Field).
Architecture of cells- Structural organization of prokaryotic and eukaryotic cells – microbial, plant and animal cells.
The ultra structure of plant cell wall, plasma membrane, nucleus, mitochondria, RES, SES, golgi apparatus, lysosome, peroxisome and . Onion tissue provides excellent cells to study under the microscope.
The main cell structures are easy to see when viewed with the microscope at medium power. For . Cells that have been fixed and stained can be studied in a conventional light microscope, while antibodies coupled to fluorescent dyes can be used to locate specific molecules in cells in a fluorescence microscope.
Living cells can be seen with phase-contrast, differential-interference-contrast, dark-field, or bright-field microscopes. To determine the field diameter of a microscope and calculate average onion epidermis cell size Pre-lab: Read Dragonfly the center of your field of view.
3. Count the number of cells lengthwise along the diameter, the widest part of the circle. Onion (x) Lab Onion Staining and Cell Size Page 3 of 3 Analysis and Conclusions: 1. Learn more about biology, paramecium, chemistry, electronics, microscopy (Microscope), Amateur Radio, Photography, Radio Astronomy, Science, Home Learning and much.