The Compound Microscope. Brightfield: Köhler Illumination
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1 Outline History of Microscopy The Magnifying Glass The Compound Microscope Brightfield: Köhler Illumination
2 Microscopy µικροσ (mikros): small σκοπειν (skopein): to observe
3 History of Microscopy Well : < BC
4 History of Microscopy Zacharias Janssen ( ): First Compound Microscope (1595)
5 History of Microscopy Cornelis Jacobszoon Drebbel ( ): 1605: Experiments with lenses, magnifications Robert Hooke ( ): First two-lens light microscope, book Micrographia, 1655
6 History of Microscopy Antoni van Leeuwenhoek ( ): High-quality lenses, 270-fold magnification, staining of specimens
7 History of Microscopy Étienne-Louis Malus ( ): Examined refraction of light and polarization George Gabriel Stokes ( ): Discovered light fluorescence, examined absorption of light
8 History of Microscopy Ernst Abbe ( ): 1866: Cooperation with Carl Zeiss, 1870: Formula for optical resolving power, 1889: Carl Zeiss foundation x = λ NA Objective + NA Condenser
9 History of Microscopy Paul Ehrlich ( ): Developed staining techniques for immunology further Hans Christian Gram ( ): Gram staining of bacteria
10 History of Microscopy August Köhler ( ): 1893: PhD degree at University of Gießen, 1894: Köhler illumination, 1900: Started at Carl Zeiss
11 History of Microscopy Frits Zernike ( ): 1930: Invents phase-contrast microscopy, 1941: Industrialization (German Wehrmacht), 1953: Nobel Prize for Physics
12 History of Microscopy Marvin Minsky (1927 ): 1955: Confocal Laser Scanning Microscope, 1956: Coined term Artificial Intelligence
13 History of Microscopy Stefan W. Hell (1962 ): 2000: First published the STED microscope, 2002: MPI Biophys. Chemistry, Göttingen 2006: German Future Award Leica TCS STED
14 The Magnifying Glass M = 250 mm f M 3
15 The Compound Microscope Sketch! Microscope optics according to DIN: Distance mounting face objective mounting face eyepiece = 160 mm (mechanical tube length)
16 The Compound Microscope Total magnification: M tot = M obj x M eyepiece Example: Objective 100x, eyepiece 10x: M tot = 1000x
17 The Compound Microscope Numerical aperture (NA) of an objective NA = n sinα
18 The Compound Microscope Numerical aperture (NA) of an objective: Useful magnification : M useful M useful, max = 1250 = ( ) x NA Resolving power (Abbe): x = λ NA Objective + NA Condenser
19 The Compound Microscope Magnification > Useful Magnif.? Empty Magnification
20 The Compound Microscope Objective markings (DIN 58878)
21 The Compound Microscope Objective markings (DIN 58878) Infinity-corrected optics: Parallel rays behind objectives. Required: Tube lens to form the real intermediate image.
22 The Compound Microscope Objectives: Optical corrections Achromats/Apochromats: Correction of chromatic aberrations
23 The Compound Microscope Objectives: Optical corrections (Semi) planachromats: flat images Feld: 18 mm 80 % Feld: 15 mm 60 % Feld: 11 mm
24 The Compound Microscope Illumination: Components Condenser Collector
25 The Compound Microscope Illumination: Beam path The collector images the lamp filament onto the aperture diaphragm.
26 The Compound Microscope Illumination: Beam path Aperture diaphragm in condenser back focus: Rays from aperture diaphragm form parallel rays behind condenser.
27 The Compound Microscope Illumination: Beam path Distance condenser specimen: field diaphragm gives sharp image in image plane.
28 Alignment for Köhler Illumination Image of Field Diaphragm not centered not centered centered opened
29 Alignment for Köhler Illumination Köhler Illumination: Effect
30 Alignment for Köhler Illumination Köhler Illumination: Components Field diaphragm Height-adjustable condenser Aperture diaphragm Essential Requirements in IVDD!!!
31 Alignment for Köhler Illumination Aperture (Condenser) Diaphragm Important for Köhler illumination When closed: contrast, resolution aperture diaphragm
32 Alignment for Köhler Illumination Aperture (Condenser) Diaphragm When closed: contrast, resolution
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