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University of Waterloo
KIN 155

Biology How Cells are Studied I Optic techniques for cellular and subcellular architecture The Light Microscopy Limit of resolution Scale of cell biology m, nm, and A Compound microscopy Types of light microscopy Brightfield microscopy basic form inexpensive and easy for color and fixed specimen and not for living species Phase-contrast microscopy phase plate good for living, unstained specimen Dark field microscopy Fluorescence microscopy fluorescent compounds exciter filter barrier filter Differential -interference -contrast microscopy (DIC) (Nomarski) polarizer analyzer Wollaston prism to produce 3-D image Confocal microscopy to produce 3-D image from a collection of optic sections Sample preparation techniques in light microscopy Fixation Cryoprotection Embedding and sectioning Staining Labeling radioisotope immunolabeling The Electron Microscopy Use a beam of electron to produce an image Two major types of electron microscopy Transmission electron microscopy (TEM) Vacuum system Electron gun Electromagnetic lenses and image formation Photographic system Sample preparation techniques in TEM microscopy Fixation Embedding, Sectioning, and poststaining Electron microscopic autoradiography Negative staining Shadowing Freeze-fracturing Freeze-etching Scanning electron microscopy (SEM): 3 D images Second electrons Sample preparation techniques in SEM microscopy Fixation Postfixation Dehydration Poststaining Mounting Coating with a layer gold or a mixture of gold and palladium. How Cells are Studied II Biochemical Techniques for Cellular and Subcelllular Functions Isolation of cells Source for the best yield fetal or neonatal tissue Disrupting the extracellular matrix and intercellular junctions Proteolytic enzymes Chelating agents Approaches to separate cell types Centrifugation Cell sorter: fluorescence-activated cell sorter What to do with a uniform population of cells For biochemical analysis For cell culture Fractionation of organelles and macromolecules Cell disruption: homogenate Centrifugation Separation by size Separation by size and shape Separation by buoyant density Cell-free system Isolation Reconstitution Chromatography Partition chromatography Column chromatography Ion-exchange chromatography Gel-filtration chromatography Affinity chromatography HPLC Electrophoresis Proteins usually have a net positive or negative charge that reflects the mixture of charged amino acids they contain. If an electric field is applied to a solution containing a protein molecules, the protein will migrate at a rate that depends onits net charge and on its size and shape SDS-PAGE SDS -mercaptoethanol Coomassie blue Silver stain Western blotting 2-D gel electrophoresis First dimension: isoelectrical focusing Second dimension: SDS-PAGE
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