Schulung Mikroskopie I engl - Hund · Outline • History of Microscopy • The Magnifying Glass...

Outline

• History of Microscopy

• The Magnifying Glass

• The Compound Microscope

we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008

• The Compound Microscope

• Brightfield: Köhler Illumination

Microscopy

µικροσµικροσµικροσµικροσ (mikros): small

σκοπειν σκοπειν σκοπειν σκοπειν (skopein): to observe


σκοπειν σκοπειν σκοπειν 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 andpolarization


• 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


1889: Carl Zeiss foundation

CondenserObjective NANAx

+=∆

λ


• Paul Ehrlich (1854 – 1915):

Developed staining techniquesfor 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


1900: Started at Carl Zeiss


• Frits Zernike (1888 – 1966):

1930: Invents phase-contrast microscopy,1941: Industrialization (German Wehrmacht),1953: Nobel Prize for Physics


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, Göttingen2006: German Future Award


Leica TCS STED

The Magnifying Glass

f

mmM

250=


M ≤ 3

The Compound Microscope

Sketch! Microscope opticsaccording to DIN: Distancemounting face objective –mounting face eyepiece= 160 mm


(mechanical tube length)


• Total magnification:

Mtot = Mobj x Meyepiece

• Example:


• Example:Objective 100x, eyepiece 10x:Mtot = 1000x


Numerical aperture (NA) of an objective


NA = n · sinαααα


Numerical aperture (NA) of an objective:

• “Useful magnification”:Museful = (500 … 1000) x NAM = 1250


Museful, max = 1250

• Resolving power (Abbe):

CondenserObjective NANAx

+=∆

λ


Magnification > Useful Magnif.?

→ “Empty” Magnification



Objective markings (DIN 58878)



Objective markings (DIN 58878)

Infinity-corrected optics:

Parallel rays behindobjectives.


objectives.

Required:Tube lens to form thereal 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

notcentered

notcentered 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 ↓


Schulung Mikroskopie I engl - Hund · Outline • History of Microscopy • The Magnifying Glass...

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