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Cell Bio Lecture 2.docx

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Biology 2382B
Robert Cumming

Light Microscopy Bright Field Light Microscopy -UsPhase Contrast Microscopyn a Differential Interference Contrast -condenser lens to focus light on mag-Changes in phase of light (DIC) Microscopy (Normarski) -Examines live ‘unstained’ cells -Interference between polarized light specimen -single cells or thin cell layers but not -examines live unstained cells -objective lens to collect light after it has passed through specimen thick tissues -defines outline of large organelles -ocular or eyepiece lens to focus -location and movement of larger such as nucleus and vacuole and image onto eye organelles in live cells provides better detail of cell edge -Typical light microscope magnification is 40 to 1000X -Only structures with a high refractive Phase Contrast Microscope index (ability to bend light) are observable -small differences in refractive index and thickness within the cell are Compound Microscope further exploited and converted into contrast visible to the eye -light moves slower in a medium with higher refractive index -cone of light generated by annular diaphragm Fluorescence Microscopy -uses a property of certain molecules to Immunofluorescence Microscopy fluoresce Two Major Limitations -a dye can be conjugated with antibodies to -Location of fluorescent dyes or fluorescent 1-Blurred image from superposition of localize any molecules of your interest in cellsotein molecules can be imaged. fluorescent images -monoclonal antibodies; a clonal cell line thatCan visualize more than one protein or Confocal Microscopys at various depths must be secretes antibodies to a specific epitope* on cell structure -Obtains image from a specific focal plane and protein excludes light from other planes by use of a Fluorescence Microscope pinhole -Antibodies are made to specific proteins (i.e.reflect excitation light on a special filter; microtubules, actin) dichroic mirror, into sample and allowing -Added to cells fixed on a slide which bind theight emitted at longer wavelength to pass specific protein they were designed to through the observer recognize -Secondary antibodies with attached fluorophores are added and bind the primary antibody -Each fluorophore has a unique excitation and emission wavelength that can be detected with appropriate filters in the microscope Dual Label Fluorescence Microscopy -Fixed (Dead) cells usedcope filter set for each flurochrome then digitally overlay images Laser Scanning Microscope -Uses a point laser light source at the excitation Fluorescent Imaging in live cells Deconvolution Microscopy -Derived from a naturally occurring protein -computational restoration method found in a jellyfish capable of bioluminescence -The protein contains a short sequence of -dyes or “FLUOROPHORES” -series of images of object taken at different amino acids (chromophore) that are capable absorb energy kicking electrons (e) into a focal planes (called a Z-stack) of fluorescing when excited with blue light higher orbital (unstable) -conventional fluorescence microscope or - confocal -mathematical processing method in -Gene was isolated and heavily modified so it - instability causes e to drop into its normal which computations for the acquired stack
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