Outline History of Microscopy The Magnifying Glass The Compound Microscope Brightfield: Köhler Illumination
Microscopy µικροσ (mikros): small σκοπειν (skopein): to observe
History of Microscopy Well : < 10000 BC
History of Microscopy Zacharias Janssen (1580 1638): First Compound Microscope (1595)
History of Microscopy Cornelis Jacobszoon Drebbel (1572 1633): 1605: Experiments with lenses, magnifications Robert Hooke (1635-1703): First two-lens light microscope, book Micrographia, 1655
History of Microscopy Antoni van Leeuwenhoek (1632 1723): High-quality lenses, 270-fold magnification, staining of specimens
History of Microscopy Étienne-Louis Malus (1755 1812): Examined refraction of light and polarization George Gabriel Stokes (1819-1903): Discovered light fluorescence, examined absorption of light
History of Microscopy Ernst Abbe (1840 1905): 1866: Cooperation with Carl Zeiss, 1870: Formula for optical resolving power, 1889: Carl Zeiss foundation x = λ NA Objective + NA Condenser
History of Microscopy Paul Ehrlich (1854 1915): Developed staining techniques for immunology further Hans Christian Gram (1853-1938): Gram staining of bacteria
History of Microscopy August Köhler (1866 1948): 1893: PhD degree at University of Gießen, 1894: Köhler illumination, 1900: Started at Carl Zeiss
History of Microscopy Frits Zernike (1888 1966): 1930: Invents phase-contrast microscopy, 1941: Industrialization (German Wehrmacht), 1953: Nobel Prize for Physics
History of Microscopy Marvin Minsky (1927 ): 1955: Confocal Laser Scanning Microscope, 1956: Coined term Artificial Intelligence
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
The Magnifying Glass M = 250 mm f M 3
The Compound Microscope Sketch! Microscope optics according to DIN: Distance mounting face objective mounting face eyepiece = 160 mm (mechanical tube length)
The Compound Microscope Total magnification: M tot = M obj x M eyepiece Example: Objective 100x, eyepiece 10x: M tot = 1000x
The Compound Microscope Numerical aperture (NA) of an objective NA = n sinα
The Compound Microscope Numerical aperture (NA) of an objective: Useful magnification : M useful M useful, max = 1250 = (500 1000) x NA Resolving power (Abbe): x = λ NA Objective + NA Condenser
The Compound Microscope Magnification > Useful Magnif.? Empty Magnification
The Compound Microscope Objective markings (DIN 58878)
The Compound Microscope Objective markings (DIN 58878) Infinity-corrected optics: Parallel rays behind objectives. Required: Tube lens to form the real intermediate image.
The Compound Microscope Objectives: Optical corrections Achromats/Apochromats: Correction of chromatic aberrations
The Compound Microscope Objectives: Optical corrections (Semi) planachromats: flat images Feld: 18 mm 80 % Feld: 15 mm 60 % Feld: 11 mm
The Compound Microscope Illumination: Components Condenser Collector
The Compound Microscope Illumination: Beam path The collector images the lamp filament onto the aperture diaphragm.
The Compound Microscope Illumination: Beam path Aperture diaphragm in condenser back focus: Rays from aperture diaphragm form parallel rays behind condenser.
The Compound Microscope Illumination: Beam path Distance condenser specimen: field diaphragm gives sharp image in image plane.
Alignment for Köhler Illumination Image of Field Diaphragm not centered not centered centered opened
Alignment for Köhler Illumination Köhler Illumination: Effect
Alignment for Köhler Illumination Köhler Illumination: Components Field diaphragm Height-adjustable condenser Aperture diaphragm Essential Requirements in IVDD!!!
Alignment for Köhler Illumination Aperture (Condenser) Diaphragm Important for Köhler illumination When closed: contrast, resolution aperture diaphragm
Alignment for Köhler Illumination Aperture (Condenser) Diaphragm When closed: contrast, resolution