The Compound Microscope

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Outline
• History of Microscopy
• The Magnifying Glass
• The Compound Microscope
• Brightfield: Köhler Illumination
we bring technologies together. optics – electronics – precision mechanics
© Helmut Hund GmbH, 2008
Microscopy
µικροσ (mikros): small
σκοπειν (skopein): to observe
History of Microscopy
• Well…:
< 10000 BC
• Zacharias Janssen (1580 – 1638):
First Compound Microscope (1595)
• Cornelis Jacobszoon Drebbel (1572 – 1633):
1605: Experiments with lenses,
magnifications
• Robert Hooke (1635 - 1703):
First two-lens light microscope,
book “Micrographia”, 1655
• Antoni van Leeuwenhoek (1632 – 1723):
High-quality lenses,
270-fold magnification,
staining of specimens
• Étienne-Louis Malus (1755 – 1812):
Examined refraction of light and
polarization
• George Gabriel Stokes (1819 - 1903):
Discovered light fluorescence,
examined absorption of light
• Ernst Abbe (1840 – 1905):
1866: Cooperation with Carl Zeiss,
1870: Formula for optical resolving power,
1889: Carl Zeiss foundation
∆x =
λ
NAObjective + NACondenser
• Paul Ehrlich (1854 – 1915):
Developed staining techniques
for immunology further
• Hans Christian Gram (1853 - 1938):
Gram staining of bacteria
• August Köhler (1866 – 1948):
1893: PhD degree at University of Gießen,
1894: Köhler illumination,
1900: Started at Carl Zeiss
• Frits Zernike (1888 – 1966):
1930: Invents phase-contrast microscopy,
1941: Industrialization (German Wehrmacht),
1953: Nobel Prize for Physics
• Marvin Minsky (1927 – ):
1955: Confocal Laser Scanning Microscope,
1956: Coined term “Artificial Intelligence”
• 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
250 mm
M=
f
M≤3
Sketch! Microscope optics
according to DIN: Distance
mounting face objective –
mounting face eyepiece
= 160 mm
(mechanical tube length)
• Total magnification:
Mtot = Mobj x Meyepiece
• Example:
Objective 100x, eyepiece 10x:
Mtot = 1000x
Numerical aperture (NA) of an objective
NA = n · sinα
α
Numerical aperture (NA) of an objective:
• “Useful magnification”:
Museful
= (500 … 1000) x NA
Museful, max = 1250
• Resolving power (Abbe):
∆x =
λ
NAObjective + NACondenser
Magnification > Useful Magnif.?
→ “Empty” Magnification
Objective markings (DIN 58878)
Objective markings (DIN 58878)
Infinity-corrected optics:
Parallel rays behind
objectives.
Required:
Tube lens to form the
real intermediate image.
Objectives: Optical corrections
• Achromats/Apochromats:
Correction of chromatic aberrations
Objectives: Optical corrections
• (Semi) planachromats: “flat” images
Feld:
18 mm
80 % Feld:
15 mm
60 % Feld:
11 mm
Illumination: Components
Condenser
Collector
Illumination: Beam path
The collector images the lamp filament onto the
aperture diaphragm.
Aperture diaphragm in condenser back focus: Rays from
aperture diaphragm form parallel rays behind condenser.
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
Köhler Illumination: Effect
Köhler Illumination: Components
• Field diaphragm
• Height-adjustable condenser
• Aperture diaphragm
“Essential Requirements” in IVDD!!!
Aperture (Condenser) Diaphragm
• Important for Köhler illumination
• When closed: contrast ↑, resolution ↓
aperture diaphragm
Aperture (Condenser) Diaphragm
• When closed: contrast ↑, resolution ↓

The Compound Microscope

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