schulung mikroskopie ii engl - hund · darkfield microscopy competitors: darkfield for less??? hund...

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”:


Obje

kt



Detection of “amplitude objects”:




Detection of “phase objects”:


Obje

kt





Epithelium cells





Epithelium cells,aperture diaphragmclosed

History

Frits Zernike (1888 – 1966):

1930: Invents phase-contrast microscopy,

1941: Industrialization (German Wehrmacht),

1953: Nobel Prize for Physics


Working Principle

Beam Path:


Working Principle

Objectives:

• Ph objective ↔ Light annulus!

• Numbering acc. to positionin (turret) condenser


in (turret) condenser

• Caution: Counting chambers!

• Caution: Fully open aperturediaphragm!

Working Principle

Control: Auxiliary Microscope

Phase annulus

Light annulus


WRONG!

CORRECT!

Working Principle


Epithelium cells

Darkfield Microscopy


What makes light rays visible?

Photo: © Dr. Jörg Haus, 2007


Shadows: Geometrical Optics


Physics: Light described as particle or plane wavefront, w/odiffraction, light rays = wavefront normals


Shadows: Small Structures, Wave Optics?


Physics: Light as spherical wavefront,Huygens‘ principle


Shadows: Small Structures, Wave Optics?


Young‘s double-slit experiment


Comparison Brightfield - Darkfield

ObjektivObjektiv


Kondensor

Präparatebene Präparatebene

Kondensor

Brightfield Darkfield


Condenser: Annulus vs. Cardioid System



H600 LL HP 100: Optical Components

Objective, SPL 100/1.25,with iris diaphragm


Condenser,NA 1.4


H600 LL HP 100: Optical Components



H600 LL HP 100: Native Blood Investigations



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


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



Novex


Hund



Novex:Objective irisdiaphragm isclosed.

No high-resolution DF


resolution DFpossible!!!

Polarization Microscopy

Physics: Light has wave properties

Unpolarizedlightwave

Polarizedlightwave

Complete


Polarization filter(Polarizer)

Completeextinction

Polarization filter(Analyzer)


Birefringence: Polarization Rotation




Glass ceramics betweencrossed polarizers,

with retardation plate


Glass ceramics betweencrossed polarizers



H 600 with polarization unit HP I:

Analyzer with compensator first-order red

Strain-free optics


Strain-free optics

Rotary table with object guide

Polarizer


Two Observation Techniques:

• Orthoscopic:Polarizer – Specimen – Analyzer,observation through eyepieces


• Conoscopic:Observation in back focal plane of objektive with retractable Bertrand lens or auxiliarymicroscope, high NA of condenser!


Two Observation Techniques:

• Conoscopic:


The Inverted Microscope

Dedicated Culture Vessels


Petri dish

Terasaki-(96-well-) plate

Culture flask

The Inverted Microscope

Applications:

• In-vitro fertilization(with micromanipulators)

• Cell-culture examinations


• 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


• 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 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 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

schulung mikroskopie ii engl - hund · darkfield microscopy competitors: darkfield for less??? hund...

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