Lecture 1--microscopy

Focus light on specimen reflect light off s urfeace

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Unformatted text preview: erty TEM slice through ER, with SEM 3D endoplasmic reticulum TEM and SEM provide different perspectives into cellular structure stereocelia tallest one true celia with microtub ules cross section of hair cells enbedded in plastid (white space) not real color becausr the pictures are black/white light microscopy, why use it?: tag different hings in cell to see specific structures can look at live cells look at 3D It would be ideal to have the resolution of TEM with the 3 dimensionality and ability to highlight particular proteins of fluorescence microscopy Super-Duper Resolution microscopy? Ability to assemble TEM slices into a 3-D image Ability to label specific proteins in a TEM Sections for Transmission Electron Microscope 3-D structures can be reconstructed from EM sections New technique for EM 3-D reconstruction: serial block face EM The tissue is placed in a block and imaged with SEM techniques from the top, a thin slice is removed and imaging is repeated New technique for EM 3-D reconstruction: Serial block face EM This technique is being used to reconstruct neurons and map their connections Takemura SY, Bharioke A, Lu Z, Nern A, Vitaladevuni S, Rivlin PK, Katz WT, Olbris DJ, Plaza SM, Winston P, Zhao T, Horne JA, Fetter RD, Takemura S, Blazek K, Chang LA, Ogundeyi O, Saunders MA, Shapiro V, Sigmund C, Rubin GM, Scheffer LK, Meinertzhagen IA, Chklovskii DB. (2013). A visual motion detection circuit suggested by Drosophila connectomics. Nature. 2013 Aug 8;500(7461):175-81 light micro scopy New technique for EM 3-D reconstruction: Serial block face EM New technique for EM 3-D reconstruction: Serial block face EM Takemura et al. (2013). Nature. 2013 Aug 8;500(7461):175-81 New technique for highlighting specific proteins in EM micrographs: Minisog cannot do immuno fluorescence because need light to excite fluorophore Minisog is a small fluorescent molecules that creates localized oxygen upon illumination that can be used to catalyze a reaction that creates an electron-dense polymer Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, et al. (2011) A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biol 9 (4): e1001041. New technique for highlighting specific proteins in EM micrographs: Minisog compone nt of gap junctions Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, et al. (2011) A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms. PLoS Biol 9 (4): e1001041. Super-Duper Resolution microscopy? Ability to assemble TEM slices into a 3-D image Ability to label specific proteins in a TEM Microscopy summary: 1. Different kinds of light microscopy can be used to visualize thin specimens (bright field, phase, interference) 2. Staining increases tissue contrast in light microscopy 3. Fluorescence microscopy relies on fluorophore excitation 4. Fluorophores can be conjugated to antibodies or expressed as fluorescent proteins 5. Confocal microscopy allows the visualization of fluorescently labeled specimens in 3-D 6. In multiphoton microscopy only a single focal point is excited 7. Super-resolution transcends the limit of light microscopy 8. TEM and SEM allow extremely high resolution imaging...
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