LabManual_Microscopy&Cells

LabManual_Microscopy&Cells - Foundations of...

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Microscopy & Cells - 1 Microscopy & Structure and Function of Living Cells THE OBJECTIVES OF THIS LABORATORY EXERCISE ARE TO: 1. Become familiar with the proper use of compound light microscopes and dissecting scopes. 2. Recognize similarities and differences of types of living cells. P ART 1: M ICROSCOPY Because the unaided eye cannot detect anything smaller than 0.1 mm (10 -4 meters) in diameter, cells, tissues and many small organisms cannot be observed directly. A light microscope extends our vision a thousand times, so that objects as small as 0.2 micrometers (2 × 10 -7 meters) in diameter can be seen. The electron microscope further extends our viewing capabilities down to 1 nanometer (10 -9 meters). At this level it is possible to see the outlines of individual proteins or nucleic acid molecules. Thanks to microscopes, our understanding of cell structure and function has greatly improved. The quality of a microscope depends upon its capacity to resolve, not magnify, objects. Magnification simply makes objects appear larger. Resolution is the capacity of an optical instrument to distinguish details. High resolution allows us to see separate points, while low resolution blurs close points into one image. Therefore, without resolving power , or the ability to distinguish detail, magnification is worthless. The theoretical limit for the resolving power of a microscope depends on the wavelength of light (the color) and a value called the numerical aperture of the lens system, times a constant (0.61). The numerical aperture is derived from a mathematical expression that relates the light delivered to the specimen by the condenser to the light entering the objective lens. If all other factors are equal, resolving power is increased by reducing the wavelength of light used. Many microscopes have blue filters because blue light has the shortest wavelength in the visible spectrum. Therefore, the minimum distance that can be resolved can be calculated by: For example, if blue light with a wavelength of 480 nanometers is used and the numerical aperture is 2, the theoretical resolving power is 146 nanometers, or 0.146 micrometers. Even with sufficient magnification and resolution, a specimen can be seen on a microscope slide only if there is sufficient contrast . Contrast is based on the differential absorption of light by parts of the specimen. Often a specimen has opaque parts or contains natural pigments (e.g. chlorophyll), but the majority of biological material is translucent. Here, visibility can be improved by using a variety of stains that bind to cellular structures and absorb light, thus providing contrast. Some stains are specific for certain chemicals and bind only to structures composed of those chemicals. Others are nonspecific and stain all structures. Wavelength
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LabManual_Microscopy&Cells - Foundations of...

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