MIT7_13f08_lab09_Protocol_DIC - MIT OpenCourseWare...

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MIT OpenCourseWare 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: .
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7.13 Fall 2008 Page | 1 Digital Optical Microscopy The invention of the microscope revolutionized biology in the 1700’s by permitting scientists to explore the world of microorganisms and details of cell structure. In the 1990s the microscope has continued to be a powerful tool for biologists. We can watch subnanometer movements of cells or single molecules in cells with digital CCD cameras, measure piconewton forces exerted by motor proteins with optical traps formed by IR lasers, reconstruct the 3D structure of a cell using powerful image analysis algorithms, follow the chemical changes in a cell using fluorescent dyes, or determine the precise location of various gene products that are tagged with antibodies, enzymes, or light emitting (fluorescent or luminescent) proteins. The dynamics of cellular responses captured in a microscope tell us about the lifestyle and life span of a structure. The purpose of the microscope module in Project Lab is to familiarize you with the operation of a state-of-the-art digital microscope. The microscope system consists of a Nikon Optiphot 2 microscope, a Photometric ImagePoint CCD camera, and a MacIntosh G3 PowerPC computer for data acquisition and image analysis. With this system, you will observe the dynamics of fluorescently tagged proteins in cultured CAD cells. The manual will illustrate how to set up a microscope for imaging with phase contrast, differential interference contrast, or fluorescence optics, and how to analyze the images using Spot or Photoshop software packages. Parts of a Microscope The microscope is divided into three parts: an illumination system, an optical system, and an image viewing system. The illumination system consists of the lamp, shutters, filters, condenser, and field diaphragm. These elements deliver and focus the light onto the specimen. The optical system consists of the objective lens and various prisms, filters, and magnifiers. They focus the image onto the oculars or camera. A CCD camera captures a digital image and the image is stored on a computer. The Microscope A microscope consists of an illumination system, an imaging system, and a detecting system. These systems focus light onto a specimen, collect light from the specimen, and record the magnified image. The specimen is mounted on a transparent glass slide and positioned on the movable stage of the microscope. Light from a bright source is focused by the collector and condenser lenses onto the specimen. Light transmitted through the specimen is collected by the objective lens and focused on the focal plane of the objective lens, creating a magnified image of the specimen. The image is then further magnified and projected by the ocular lens (eyepiece) onto a detector, either the human eye, a piece of photographic film, or imaging chip of a CCD camera. The condenser diaphragm and
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MIT7_13f08_lab09_Protocol_DIC - MIT OpenCourseWare...

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