BIOL3520Microscopy - One thing about cells is they are very...

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Unformatted text preview: One thing about cells is they are very small. Overview I. Become familiar with basic principles of microscopy and use of the compound light microscope II. Learn how to perform measurements under the microscope Use statistics to account for variability and error, n draw more meaningful conclusions Understand plate magnification n n III. Examine prepared specimens IV. Advanced Microscopy presentation (~2 hr after start of lab) Technology helps drive cellular investigation. Advances in microscopy ca. 1839 ca. 1870 ca. 1650 ca. 1890 Student’s microscope (France) Hooke Leeuwenhoek (simple—not compound) Compound Light Mhe compound light microscope icroscope T was a groundbreaking tool and remains a workhorse instrument Increased magnification power and resolution Different methods of visualization Magnify based on different characteristics using different approaches Specialized applications Compound Light Images Fat-Stained Adipose Tissue Mammalian Taste Buds Volvox Pennaria Hydrozoa Inverted microscope View specimen from below Objective lens located below Light source, etc. located above n n Easier to see things that settle to bottom of container or microscope slide Easier access Switch between specimens Manipulate a specimen while viewing Olympus Corp. Conventional fluorescence microscope An “excitation” wavelength of light strikes the specimen Bathes the entire specimen in light = “widefield” n Light-active molecules in the specimen are excited and fluoresce a certain wavelength This “emission” wavelength is passed to the viewer while other wavelengths are blocked n Uses Visualize naturally-fluorescing compounds (e.g. chlorophyll) Use molecular labels to target, identify features Conventional fluorescence microscope Camera (optional) Excitation light source Conventional fluorescence microscopy n Diatoms: single-celled organisms that deposit cell walls of silica Phytoplankton: aquatic, photosynthetic n PDMPO: stains silica only while it’s being formed Can examine, monitor cell wall formation over time n Chlorophyll: fluoresces red Normal Normal light with fluorescence fluorescence Mark Demarest, UC Santa Barbara Conventional fluorescence microscopy Mark Demarest, UC Santa Barbara Conventional fluorescence microscopy Mark Demarest, UC Santa Barbara Conventional fluorescence microscopy Antenna What is this? Diatom A zooplankter eating a diatom Siliceous teeth Mark Demarest, UC Santa Barbara Conventional fluorescence microscopy Zooplankter, Zooplankter, normal light FDA stained with FDA: fluorescein diacetate; cleaved by esterases to release the fluorescent molecule fluorescein Mark Demarest, UC Santa Barbara Conventional fluorescence microscopy Endothelial cell stained for tubulin (green), mitochondria (red), and DNA (blue). Invitrogen Bacteria stained with SYTOX-Blue: only labels dead cells. Invitrogen Moth sperm: mitochondria are red, DNA blue. Laura K. Garvey, U. of Connecticut Urothelial cell nuclei stained with DAPI and marked using FISH (DNA sequencespecific probing technique). Bioview Bovine pulmonary artery endothelial cells: nuclei (blue), microtubules (green), actin filaments (red). National Institutes of Health Confocal fluorescence microscope n n n n Fluorescent light is narrowly focused on one small spot Interfering light emitted from out-of focus areas is blocked by another focusing mechanism Result: No out of focus areas visible Improved resolution: no “light wash” interference Point of focus must be rapidly scanned over surface to re-create entire image The focal depth can be moved up and down to visualize specific planes Can reconstruct into a 3-D image Confocal fluorescence microscopy Conventional Confocal Nikon Inc. Confocal fluorescence microscopy Natural (1) and Bnfad2 transgenic line 84a-B cotton cotyledon samples stained with the neutral lipid-specific stain Nile Red to reveal oil bodies. Chapman et al. 2008 Electron microscopes Use a beam of electrons instead of light for imaging Focused using magnetic fields instead of glass lenses Much better resolving power: electrons far smaller than any λ n Transmission electron microscope (TEM): electrons passed through specimen Analogous to compound light microscope Provides a two-dimensional image of a thin section n Scanning electron microscope (SEM): electrons bounced off surface of specimen Analogous to a dissecting microscope Provides a three-dimensional image of surface TEM TEM Multiple synapses in rat cortex. Anna Klintsova Chloroplast grana stacks. A. Pastor Center for Advanced Microscopy, Michigan State U. TEM Insect epithelial cells. A. Pastor Center for Advanced Microscopy, Michigan State U. Portion of a cotton cotyledon cell, showing oil and protein bodies (bar = 1µm). Chapman et al. 2008 SEM SEM Various diatoms (fresh & dissolving). James Weaver and Mark Demarest, UC Santa Barbara Centre for Microscopy and Microanalysis, University of Queensland, Australia Diatom Blood cells Diatom. unknown source Silicoflagellate. James Weaver and Mark Demarest, UCSB Diatom. unknown source SEM Pollen. Dartmouth Electron Microscope Facility Sources unknown SEM: bacteria On the end of a pin With DNA Scanning Probe Microscopes n A probe scans and interacts with the surface of a specimen on the atomic level The probe reports findings to a computer Topography, atomic composition, etc. reconstructed JEOL Ltd. SPM Atomic force microscope image of the surface and sub-surface of fresh articular cartilage from a bovine humeral head. Daniel J. Müller, Ueli Aebi and Andreas Engel, Maurice Müller Institute, Basel, Switzerland SPM Atomic force microscope topographs of purple membrane from Halobacterium salinarium. Daniel J. Müller, Ueli Aebi and Andreas Engel, Maurice Müller Institute, Basel, Switzerland ...
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This note was uploaded on 04/10/2011 for the course BIOL 3520 taught by Professor Staff during the Spring '11 term at North Texas.

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