• Document: Practical Introduction to Light Microscopy
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Practical Introduction to Light Microscopy This presentation has been put together as a common effort of Urs Ziegler, Anne Greet Bittermann, Mathias Hoechli. Many pages are copied from Internet web pages or from presentations given by Leica, Zeiss and other companies. Please browse the internet to learn interactively all about optics (i.e. microscopy.fsu.edu/primer/ ). For questions & registration please contact www.zmb.unizh.ch . Up-right Microscope Olympus Inverted Microscope Leica The inverted microscope stand allows the obser- vation of living cells in culture dishes from under- neath. An environmental box can be mount- ed to maintain temperature and CO2 concentration. The parts of the microscope • Detector (PMT, CCD) • Objective (±Immersion medium) • Specimen (Cover glas) • Light conditioning system – Köhler illumination – Phase ring – Wollaston prism and polarizers – Filter cubes (for fluorescence) • Light source (Halogen, XBO, ...) specimen types • Bulk specimens (autofluorescence, total reflection) in any kind of holder (CLSM only). i.e. teeth, casts, scaffolds, bone • Thin specimens: tissue sections, cell cultures (fluorescence-labeled, autofluorescent) on slides • Live cell imaging of native (i.e. DIC, Ph2) or transfected (i.e. GFP) cell layers in suitable chamber environments Fixation in light microscopy Fresh Paraformaldehyde solution • stopps cellular dynamics • preserve cells during preparation by crosslinking of cellular components. Solvents (Methanol, Acetone, ...) do not cross link anything but make holes into membranes instead -> no good structure preservation. Glutaraldehyde shows autofluorescence. Formaldehyde preparations Formalin = 37% formaldehyde plus ca. 10% methanol Commercially available formaldehyde solutions without methanol is unstable in time: forms polymers especially at 4°C Paraformaldehyde is the solid polymer -> use this to prepare fresh formaldehyde solutions and use them right away the light microscopic specimen ° cover glas (0,17 mm) ° sample surrounded by embedding medium (might contain anti-bleach agent) ° glas slide specimen devices for inverted light microscopes -> cover glass bottom allows also use of DIC and high resolution immersion objectives • coverslip chamber (with 1-4 chambers) • full glass bottom dish • culture dish with cover glass insert (w & w/o finder) specimen devices for inverted light microscopes -> plastic bottom dishes or plated allows use of long distance air objectives only, DIC is not possible. Some plastics show autofloures- cence. -> multi-well plates: multipe table positions can be programmed to follow cells under different conditions during the same time intervall systems for live cell microscopy: the Ludin chamber • fast transfer and mounting of the cultured coverslip • bottom cover glass allows for immersion objectives! • built-in liquid and gas perfusion lines • closed and open/covered configurations • 16 mm diameter viewable area. • designed for easy access also for micromanipulation, microinjection and electrophysiology systems for short term live cell microscopy: ibidi technology cells -> sterile plastic devices for cell culture -> plastic bottom foil has the medium reservoir same optical properties as 0,17mm cover glass medium exchange 3) -> perfusion designs possible -> gas exchange via bottom foil possible -> small volume tunnel (100 µl or 30 µl) -> multi tunnel slides available (6-tunnel-slides) specimen devices for low volume analysis (inverted LM) features: -> 18 micro-well design (5 mm diameter & 30 µl volume)

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