Outline
• Phase Contrast – why?
• History
• Working Principle
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
• Working Principle
• Dark-Field Microscopy
• Polarization Microscopy
• The Inverted Microscope
Phase Contrast – Why?
• Brightfield Microscope:
Detection of “amplitude objects”:
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Obje
kt
Phase Contrast – Why?
• Brightfield Microscope:
Detection of “amplitude objects”:
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Phase Contrast – Why?
• Brightfield Microscope:
Detection of “phase objects”:
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Obje
kt
Phase Contrast – Why?
• Brightfield Microscope:
Detection of “phase objects”:
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Epithelium cells
Phase Contrast – Why?
• Brightfield Microscope:
Detection of “phase objects”:
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Epithelium cells,aperture diaphragmclosed
History
Frits Zernike (1888 – 1966):
1930: Invents phase-contrast microscopy,
1941: Industrialization (German Wehrmacht),
1953: Nobel Prize for Physics
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Working Principle
Beam Path:
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Working Principle
Objectives:
• Ph objective ↔ Light annulus!
• Numbering acc. to positionin (turret) condenser
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in (turret) condenser
• Caution: Counting chambers!
• Caution: Fully open aperturediaphragm!
Working Principle
Control: Auxiliary Microscope
Phase annulus
Light annulus
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WRONG!
CORRECT!
Working Principle
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Epithelium cells
Working Principle
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Epithelium cells
Darkfield Microscopy
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
What makes light rays visible?
Photo: © Dr. Jörg Haus, 2007
Darkfield Microscopy
Shadows: Geometrical Optics
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Physics: Light described as particle or plane wavefront, w/odiffraction, light rays = wavefront normals
Darkfield Microscopy
Shadows: Small Structures, Wave Optics?
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Physics: Light as spherical wavefront,Huygens‘ principle
Darkfield Microscopy
Shadows: Small Structures, Wave Optics?
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Young‘s double-slit experiment
Darkfield Microscopy
Comparison Brightfield - Darkfield
ObjektivObjektiv
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Kondensor
Präparatebene Präparatebene
Kondensor
Brightfield Darkfield
Darkfield Microscopy
Condenser: Annulus vs. Cardioid System
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Darkfield Microscopy
H600 LL HP 100: Optical Components
Objective, SPL 100/1.25,with iris diaphragm
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Condenser,NA 1.4
Darkfield Microscopy
H600 LL HP 100: Optical Components
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Darkfield Microscopy
H600 LL HP 100: Native Blood Investigations
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Darkfield Microscopy
Competitors: Darkfield for Less???
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Darkfield Microscopy
Competitors: Darkfield for Less???
Hund H600 LL HP 100 Novex B mit DF
Tube (Standard) binocular trinocular, 100/100
Eyepieces WF 10x/18 WF 10x/(n. a.)
Objectives Achro 10/0.25 Plan 4/0.10
SPL 100/1.25 – 0.60 Oel Plan 10/0.25
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
SPL 100/1.25 – 0.60 Oel Plan 10/0.25
Plan S40/0.65
Plan 100/1.25 Oel
Darkfield Condenser NA 1.4 NA 1.2
Illumination attached to stand, external,
100 W halogen 150 W halogen
Price incl. VAT [€] 4474,40 1649,95
Darkfield Microscopy
Competitors: Darkfield for Less???
Novex
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Hund
Darkfield Microscopy
Competitors: Darkfield for Less???
Novex:Objective irisdiaphragm isclosed.
No high-resolution DF
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
resolution DFpossible!!!
Polarization Microscopy
Physics: Light has wave properties
Unpolarizedlightwave
Polarizedlightwave
Complete
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Polarization filter(Polarizer)
Completeextinction
Polarization filter(Analyzer)
Polarization Microscopy
Physics: Light has wave properties
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
Photo: © Dr. Jörg Haus, 2008
Polarization Microscopy
Birefringence: Polarization Rotation
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Polarization Microscopy
Birefringence: Polarization Rotation
Glass ceramics betweencrossed polarizers,
with retardation plate
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Glass ceramics betweencrossed polarizers
Polarization Microscopy
Birefringence: Polarization Rotation
H 600 with polarization unit HP I:
Analyzer with compensator first-order red
Strain-free optics
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Strain-free optics
Rotary table with object guide
Polarizer
Polarization Microscopy
Two Observation Techniques:
• Orthoscopic:Polarizer – Specimen – Analyzer,observation through eyepieces
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
• Conoscopic:Observation in back focal plane of objektive with retractable Bertrand lens or auxiliarymicroscope, high NA of condenser!
Polarization Microscopy
Two Observation Techniques:
• Conoscopic:
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The Inverted Microscope
Dedicated Culture Vessels
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Petri dish
Terasaki-(96-well-) plate
Culture flask
The Inverted Microscope
Applications:
• In-vitro fertilization(with micromanipulators)
• Cell-culture examinations
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• Cell-culture examinations
• Examinations “frombelow”
Wilovert Standard HF 40
Specifications:
• Lamp 12 V / 30 W
• Condenser NA 0.25 w/ aperture diaphragm
• Objectives with large
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
• Objectives with largefree working distance:
A 4/0.10A 10/0.25SPL 20/0.50 (LD)SPL 40/0.60 (LD),correction mount
Wilovert Standard PH 40
Specifications:
• Lamp 12 V / 30 W
• Combin. cond. NA 0.25w/ aperture diaphragm
• Objectives with large
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
• Objectives with largefree working distance:A 4/0.10A PH1 10/0.25SPL PH2 20/0.50 (LD)SPL PH2 40/0.60 (LD),correction mount
Wilovert AFL 40 PH
Additional specifications:
• HBO 100 W
• Filter slide
• Object guide
• Objectives with large
we bring technologies together. optics – electronics – precision mechanics © Helmut Hund GmbH, 2008
• Objectives with largefree working distance:AC 4/0.10A PH1 10/0.25SPL 20/0.35 (LD)SPL PH2 20/0.50 (LD)SPL PH2 40/0.60 (LD),correction mount