Elektronen-Mikroskopie PDF

(Elektronen) - Mikroskopie Otto Berninghausen 1. Geschichte der Mikroskopie 2. Grundlagen der Lichtmikroskopie 3. Fluoreszenzmikroskopie § High-Res Mikroskopie 4. Elektronenmikroskopie § Konventionelle TEM § 3D EM Antony van Leeuwenhoek, (1632 – 1723). Member Royal...

(Elektronen) - Mikroskopie Otto Berninghausen 1. Geschichte der Mikroskopie 2. Grundlagen der Lichtmikroskopie 3. Fluoreszenzmikroskopie § High-Res Mikroskopie 4. Elektronenmikroskopie § Konventionelle TEM § 3D EM Antony van Leeuwenhoek, (1632 – 1723). Member Royal Society 1680 – 1674 Infusoria – 1676 Bacteria – 1677 Sperm cells – 1682 Striated muscle 3  Early Microscopes Early 1600s Late 1600s Early 1800s Mid 1800s Late 1800s Early 1900s Morphologische Beobachtungen als Wissensgewinn Cell Theory Mikroskop Lichtquelle Kondensor Probenhalter Objektiv Okular Lichtmikroskopie  Mikroskop Objektive Numerische Apertur α α α Working distance α α α NA = n sin(α) NA: Numerical aperture n: medium refractive index α: one-half of the objective angular aperture Light Transmitting Through the Sample: Brightfield Transmitted Light Microscopy Adrenal gland Leaf section Fluoreszenz Mikroskopie Fluoreszenzfarbstoffe für sensitive und spezifische Markierungen  Fluorescence Jablonski diagram: 3 levels system Higher vibrational states First excited Singlet state Triplet state Ground Singlet state Absorption spectra Fluorescence spectra Stokes shift Fluoreszenzmikroskopie (Weitfeld) mit dichroitischen Filtern  Fluorescent Molecules Fluorescent Molecules DAPI GFP Immunohistochemistrie mit fluoreszierenden Antikörpern  Multi-labelling Excitation and Emission Spectra of Fluorescent Dyes DNA microtubules centromeres Live-Cell Fluorescence Microscopy  Lichtwellen-Interferenz Light waves travel by slightly different routes, so that they interfere with one another and cause optical diffraction effects. At high magnification, an edge appears as a series of lines, a point of light as a concentric pattern  Spatial resolution: Flourescence Numerical aperture α α α Working distance α α α NA = n sin(α) NA: Numerical aperture n: medium refractive index àBarely resolved àResolved α: one-half of the objective angular aperture Rayleigh criterion: Two adjacent object points are defined as being resolved when the central diffraction spot (Airy disk) of one point Radius Airy disk coincides with the first diffraction minimum of the other point in the image plane λ rAiry = 0.61 NA For example: for NA= 1.3; λ= 546 nm Lateral resolution= 0.61λ/NA= 260 nm.  Auflösung Abbe’sche Auflösungs Formel: nx,y = λ/2NA D = 0.2 µm Numerical Aperture NA = n sin(θ) n - refractive index of the imaging medium ( air, oil) θ - aperture angle  Point-spread-function n Messung der PSF mittels eines 175nm-beads  Verbesserung der Point-Spread Funktion (PSF) Basis: Eine höhere numerische Apertur, eine kürzere Wellenlänge, ein höherer Brechungsindex Bessere PSF in Z- Ebene (axial): Konfokale und Light-Sheet Mikroskopie Fluorescence microscopy – confocal  Konfokales Mikroskop das „pinhole”... die konfokale Lochblende WF CF Konfokale Laser-Scanning-Mikroskopie Konfokale Laser-Scanning-Mikroskopie Laser Scanning Confocal Microscope Fluorescence microscopy – confocal focal planes Scanning results in 3D stack of individual 2D sections (z-stack) Confocal microscopy – 3D rendering, reconstruction and analysis developing fly eye photorecoptors glia Christophe Jung, BioSys M Light-Sheet Fluorescence Microscopy Light-Sheet Fluorescence Microscopy Photo-Damage Comparison  Light-Sheet Fluorescence Microscopy Vertical resolution +++ +++ Speed - +++ Photo damage - +++  High-Resolution Fluoreszenzmikroskopie Überwinden der Abbe’schen Auflösungsgrenze STED / PALM / STORM STimulated Emission Depletion PhotoActivatable Localization Microscopy Stochastic Optical Reconstruction Microscopy  Resolution Abbe diffraction limit Abbe Resolutionx,y = λ/2NA D = 0.2 µm Numerical Aperture NA = n sin(θ) n - refractive index of the imaging medium ( air, oil) θ - aperture angle (1,4 in the best case)  Super Resolution Microscopy / STED Stimulated Emission Depletion (STED) The nobel price in Chemistry 2014: E. Betzig (PALM), S. Hell (STED) and W. Moerner (PALM)  Photoactivated Localization Microscopy (PALM) / Stochastic Optical Reconstruction Microscopy (STORM)  Super Resolution Microscopy / PALM Photoactivated Localization Microscopy (PALM) / Stochastic Optical Reconstruction Microscopy (STORM) Confocal PALM Resolution: ~30nm  PALM Photo-activated localization microscopy Auflösunglimit von 200nm auf