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BCOR103 Cell Imaging

by: Simran

BCOR103 Cell Imaging BCOR 103

Marketplace > University of Vermont > Biology > BCOR 103 > BCOR103 Cell Imaging

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About this Document

A summary of the first lecture of Molecular & Cell Biology.
Molecular & Cell Biology
Dr Nathan Jebbett, Dr Doug Johnson, Dr Greg Gilmartin
Class Notes
molecular biology, Cell Biology, Biology, premed
25 ?




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This 2 page Class Notes was uploaded by Simran on Tuesday January 19, 2016. The Class Notes belongs to BCOR 103 at University of Vermont taught by Dr Nathan Jebbett, Dr Doug Johnson, Dr Greg Gilmartin in Spring 2016. Since its upload, it has received 181 views. For similar materials see Molecular & Cell Biology in Biology at University of Vermont.


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Date Created: 01/19/16
CellImaging TERMSTOKNOW Contrast – difference in appearance/light absorbed between neighboring objects or parts of an object Magnification – degree to which apparent size of an object can be expanded vs. naked eye Resolution – how well closely spaced objects can be seen as separate MICROSCOPY  Source of illumination e.g. visible light (400-700nm), UV light (200-300nm), electrons (0.004nm)  System of lenses to focus illumination on the specimen e.g. glass lenses, electromagnetic lenses  Detector e.g. eye, photographic film, electronic detector (video camera)  Contrast agent (in some cases) STANDARD LIGHT MICROSCOPE 1. Condenser lens gathers diffuse light rays to illuminate specimen 2. Specimen may absorb, refract, or have no effect on incident light 3. Objective lens collects both light that has and has not been altered by specimen, altered rays are focused as an enlarged image in the tube 4. Image formed by objective lens is used as object by ocular lens creating a further magnified image which is focused by the lens of the observer’s retina in the eye PROPERTIES OF LIGHT AND IMAGE FORMATION As light passes through an object, Interference patterns Magnification cannot overcome the resolution limit waves can be in phase producing a are produced at the ( magnification ≠ resolution) bright spot or out of phase producing a edges of objects due to dim or dark spot, the image is then the wave nature of light constructed from this digital pattern causing blurred edges at the micro scale At sizes comparable to wavelengths of light, individual points will appear as expanded disks and when those points are closer together than the expansion of the individual disks the points cannot be resolved and appear as a single object RESOLUTION d=0.61λ/nsinαwheredis min.distance twopointsmustbe separatedbytobe resolved,λiswavelengthoflight (527nm=white light), nisrefractiveindexofmediumbetween objectivelensandspecimen,and α isequalto½ angleofcone oflightentering objectivelens. nsinα =numericalaperture(NA),measureslightgathering propertiesofobjective lens(quality) ; for lensactingin air bestpossible NA=1, foroil lensbestpossible NA=1.5 Limitofresolution isfoundwhen we substitute theshortestvisible wavelength andhighestpossible NA; foralight microscopethisis200nmwhichissufficienttoresolve organelles. TYPES OF LIGHT MICROSCOPY  Bright-field - Simple transmission of light through specimen - Limitation = living tissue or cells show little variation in optical density thus little contrast - Cells/tissues must be fixed, sectioned, and stained e.g. Hematoxalin (binds [-] charges such as DNA of nuclei) & Eosin (binds [+] charges such as cytoplasmic/extracellular components)  Interference - Can give an image of a live unstained cell through phase- contrast; light passing through the specimen is altered to significantly out of phase, setting up light and dark areas which reflect different densities and “edges” of the specimen  Fluorescence - Chemical compounds absorb UV radiation and release energy in the visible wavelength range; a fluorochrome can beincorporatedintothecellandexposedtoUVradiation to give high contrast to the stained object (appears colored against black background) in presence of Ca ions - Uses high intensity UV light source and dichroic mirror which reflects UV to specimen while allowing emitted light to pass through and be collected forming an image  Immunofluorescence - Antibody is produced against cellular component of interest, cell/tissue is fixed and permeablized, primary antibody is applied to attach to component and visualized using a secondary antibody coupled to fluorochrome - GFP is a small protein that can be coupled to component of interest by recombinant DNA techniques; resulting chimericgene/protein can beincorporatedintocell whereit will behave as target protein would in normal cell  ConfocalScanning - High intensity light source such as laser is used, incident light is limited to pinhole and focused by lens one point in one focal plane of the specimen and scanned across the specimen illuminating neighboring points, a secondpinhole brings the light emitted by the specimen to a focal point within the scope so only light from the studied plane reaches the detector and creates the image.


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