Material Chemistry
Contents
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Optical Microscopy
Historical development
Lens aberrations
Transmission / Reflection Microscopy
Dark-field Microscopy
Differential Interference Contrast Microscopy
4B-Microscopy
Fluorescence Microscopy
Confocal Microscopy
Nearfield Scanning Optical Microscopy
Electron Microscopy
Atomic Force Microscopy
Invention of Optical Microscopes
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Earliest version - Single lens microscope
Antonie van Leuuwenhoeck, 1660
magnification 275-fold, 1.3 :m
Improvement of Optical Microscopes
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Compound Microscope - two lens design
Improved Design,
John Yarwell, 1680
Begin of todays optical microscopy
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Ernst Abbe
Ernst Abbe together with Carl Zeiss
published a paper in 1877 defining the
physical laws that determined resolving
distance of an objective. Known as Abbe’s
Law
“minimum resolving distance (d) is related to
the wavelength of light (8) divided by the
Numeric Aperture, which is proportional to
the angle of the light cone (2) formed by a
point on the object, to the objective”.
Abbe
Diffraction limit
Begin of todays optical microscopy
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Carl Zeiss
Abbe and Zeiss developed oil immersion
systems by making oils that matched the
refractive index of glass. Thus they were able
to make the Numeric Aperture (N.A.) to the
maximum of 1.4 allowing light microscopes
to resolve two points distanced only 0.2
microns apart (the theoretical maximum
resolution of visible light microscopes).
Leitz was also making microscope at this
time.
Carl Zeiss 1816-1888
Immersion Objectives
Begin of todays optical microscopy
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Otto Schott
Dr Otto Schott formulated glass lenses that
color-corrected objectives and produced the first
“apochromatic” objectives in 1886.
Chromatic Aberation-free Objectives
Optical Imaging
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Refraction of light / Snellius law
Index of Refraction depends on material
! different speeed of light
Application: Curved surfaces
"
$
F
$
"
f
Optical Lenses
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Properties
f
p
f
2
f
q
Optical Lenses
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Limitations - Abberations
Optical Lenses
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Aberrations
F1
F2
Chromatic
aberration
F1
Corrected lens
Spherical
aberration
Optical Lenses
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Astigmatism
Optical Lense Aberrations
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Summary
Monochromatic Aberrations (dependent on form of lens)
Spherical aberration
Coma
Astigmatism
Flatness of field
Distortion
Chromatic Aberrations (dependent on wavelength of light)
Longitudinal aberration
Lateral aberration
Light path in optical microscope
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Optical Microscope
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Basic setup
Optical Microscopes
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Transmission Reflection Setup
Optical Microscopes
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Upright and Inverted Setup
Optical Microscopes
Reparation of real images of light source and object
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Limit for smallest resolvable distance d
between 2 points is (Rayleigh criterion):
A
2
Light cone
NA=n(sin2)
Thus high NUMERICAL APERTURE is
critical for high magnification
(n=refractive index)
Optical Microscopy
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Numerical Aperture
Typical Objectives
Cheap:
Expensive:
Oil Immersion:
NA 0,1 Resolution 2,75 :m
NA 0,65 Resolution 0,42 :m
NA 1,4 Resolution 0,20 :m = 200 nm
Optical Microscopy
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Magnification vs. Resolution
Objectives
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Numerical aperture, resolution and working distance
< The wider the receiving angle of the lens the greater its resolving power.
< The higher the NA the shorter the working distance.
Objectives
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Immersion and cover glass thickness
Objectives
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Nomenclature
F - Infinity
corrected
PLAN-APO-40X1.30 N.A. 160/0.22
Flat field Apochromat Magnification Numerical Tube Coverglass
Aperture Length Thickness
Factor
Optical Industry in Germany
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... quite a time ago
Tubusl. 160 mm
4.0 mm
Apert 0.95
C. Zeiss Jena
No. 21
Apochromat Zeiss von 1886
vernickeltes und zaponiertes Messing, gebläuter Stahl, in signierter Messingdose.
Mehrzeilig signiertes und nummeriertes Objektiv mit Korrektur
Illumination of Objects
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Bright field
Dark field
Full aperture
illuminated
Central part of
light cone is
blocked
Illumination of Object
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Dark-Field
A central obstruction blocks the central
cone.
The sample is only illuminated by the
marginal rays.
These marginal rays must be at angles too
large for the objective lens to collect.
Only light scattered by the object is
collected by the lens. (Tyndall effect)
No staining of objects required.
Imaging Techniques
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Dark field illumination
Not absorption but refractive index
changes are imaged.
This causes that mostly the outer
structure is well received,
however, inner structures are
often invisible.
Imaging Techniques
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Phase Contrast Microscopy
Principle
1) Blocking using a ring aperature
before illumination of the object
2) Phase rotation by 8/2 plate of
the object light
3) Overlapping of objecte light
and illuminating light
Transmission
Phase Contrast
Phasenkontrast-Mikroskopie
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Phasenshift näherungsweise proportional der Dichte
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Phasen-Kontrast beim Röntgen
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Rattenherz
Phase Contrast Microscopy
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Principle
2 apertures in close proximity
first diffraction orders are far from
zeroth order
! strong disturbance on main
image (visible)
2 apertures more separated
first diffraction orders are close to
zeroth order
! weaker disturbance off main
image (lower visibility)
Differential Interference Contrast (DIC)
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Taking advantage of polarized light and optical birefringence
DIC
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Imaging Techniques
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Differential Interference Contrast (DIC) Microscopy
Imaging techniques
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Differential Interference Microscopy
Electron Microscopy
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DIC
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Flourescence Microscopy
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Principle
Fluorescence Microscopy
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Not Diffraction limited
Fluorophores
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Chemical Structures
Confocal Microscopy
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Principle setup
Principle
1) Point like illumination of the
object by means of a first aperture.
Focusing in three dimensions
2) Detection of object beams from
focal points different in lateral AND
vertical position at second
aperture. All other layers
contribute less than proportional
3) Imaging by 3D-Scanning
Confocal Microscopy
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Scanning System
Confocal Microscopy
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Examples
Paramecium labeled with
an anti-tubulin-antibody
showing thousands of cilia
and internal microtubular
structures.
Whole mount of Zebra Fish larva
stained with Acridine Orange,
Evans Blue and Eosin.
Comparison of Fluorescence and Confocal Microscopy
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One layer out of an image stack
Mitotic Cell
Imaging conditions
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Wide field, confocal, multiphoton
4B-Mikroskopie
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Fluoreszenzspektroskopie mit optimaler Auflösung
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Confocal vs. 4B
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Golgi Apparat
Confocal Microscopy
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Resolution
The Nyquist Theorem
Nyquist theory describes the sampling frequency (f) required to
represent the true identity of the sample, i.e., how many times must
you sample an image to know that your sample truly represents the
image?
In other words to capture the periodic components of frequency f in
a signal we need to sample at least 2f times
Nyquist claimed that the rate was 2f. It has been determined that in
reality the rate is 2.3f - in essence you must sample at least 2 times
the highest frequency.
For example in audio, to capture the 22 kHz in the digitized signal,
we need to sample at least 44.1 kHz (unless of course you can’t
hear 22kHz and then you don’t need 44.1 kHz!!!!)
Doing ‘fast’ images is nonsense !
Microscopes and their resolution
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Normal Optical Microscope
Resolution 200 nm
Phase Contrast Microscopy Enhanced Contrast
DIC-Microscopy
Enhanced Contrast, Height measurement
Confocal Microscopy
3D-Images, Resolution 150 nm
4Pi-Microscopy
Resolution 70 nm
NSOM
Resolution 20 nm
AFM
Resolution 10 nm, Force Measurement
Fluorescence-Technique
Resolution like carrier Microscope
extreme contrast,
single atoms/molecules emitting
NSMM
Resolution 200 nm
Measurement of dielectric constant