Microscopy is about a combination of resolution (seeing smaller and smaller things), and contrast (seeing what you want to see).

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Microscopy is about a combination of resolution
(seeing smaller and smaller things), and contrast
(seeing what you want to see). Both aspects have
recently seen great advancements, with the advent
of single molecule studies (in dilute solutions)
and super-resolution, beating the Abbe-limit.
Here, we will treat both aspects, starting with
contrast agents that are either added or that
occur naturally.
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• Contrast
• - Polarization, birefringence
• Fluorescence lifetime
• Fluorescence transfer
• Resolution/Contrast
• Two Photon Microscopy
• Single plane illumination
• Resolution
• Stimulated Emission Depletion
• Photo-activated localization
• Structured Illumination
• Total internal reflectance
• Scanning near field

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Polarization I
property of light waves, describes orientation of
oscillations
http//www.ifsc.usp.br/lavfis/BancoApostilasImage
ns/ApEfFotoelastico/photoelasticity.pdf
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Polarization microscopy
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Polarization II
http//www.ifsc.usp.br/lavfis/BancoApostilasImage
ns/ApEfFotoelastico/photoelasticity.pdf
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Birefringence
ellipticity f(d) (n n ) d/l
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Results in conoscopic figures
Problem!
nematic liquid crystals
cholesteric liquid crystals
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Twisted liquid crystal cell polarization switch
light switch - can be used as a filter
(Kerr effect)
Used in flat screens (TV, notebooks, beamers) ,
TFT LCD thin film transistor liquid crystal
display
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Birefringence image is INDEPENDENT of orientation
of optical axis
White 0.3nm retardance
Microtubule aster image
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movie
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Fluorescence
Fluorescence principle
Vibrational states
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Light basically interacts with the electrons in a
given material. The photons making up the light
in Quantum mechanics can be absorbed, if there is
an electron state at the energy corresponding to
the absorbed energy of the photon. Once a photon
is absorbed and the electrons are excited, they
can either relax via collisions and vibrations or
by emitting another photon. In case the electron
relaxes before emitting another photon, there is
fluorescence.
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Natural proteins for fluorescence studies
Green fluorescent protein (GFP)
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Fluorescence lifetime microscopy (FLIM)
Lifetime image
A. GFP-tagged protein B. YFP- tagged protein C
D. Both GFP and YFP tagged proteins. The colour
bars show the calibration of fluorescence
lifetime from approx 2.1ns (red) to 3.0ns (dark
blue).
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Measuring viscosities using FLIM
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Measuring temperatures using FLIM
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Forster resonance energy transfer (FRET)
Overlap of emission and absorption spectra of
flouorophores can be used to obtain information
on their distance
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FRET measures distance changes in the nanometer
scale, a relevant length scale for many
biomolecules.
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Histone phosphorylation (specific for serine 28
on histone 3) in living HeLa cells undergoing
cell division. Red signifies high FRET (and high
phosphorylation levels), blue signifies low FRET
(and low phosphorylation levels), and green is
intermediate. The reporter displays a rapid
increase in FRET 5-15 minutes after breakdown of
the nuclear envelope.
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Functional studies using FRET on single molecules
in real time
Time record of folding and unfolding of an RNA
molecule hairpin ribozyme. We attach the donor
(green) and acceptor (red) dyes to the RNA so
that the folded state has high FRET and the
unfolded state has low FRET. We can see this
beautiful two-state fluctuations in FRET values
as a function of time
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Two photon fluorescence microscopy
Principle of 2 photon fluorescence
Radiationless decay
fluoro
2 NIR photons are absorbed simultaneously
weakest absorption
Best penetration near 1000nm
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Typical two photon fluorescence setup
High intensity required !
2 hv excit
Fluorescence detection
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Selective plane illumination microscopy (SPIM)
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Scanning SPIM (DLSM)
Allows for faster scanning and induces less
photons to the sample, since only a single line
is illuminated. Long-time imaging becomes
possible.
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Movie of Zebrafish embryo nuclei
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Stimulated emission depletion (STED) microscopy
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Typical setup
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Photo-activated localization microscopy (PALM)
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The principle of PALM
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The principle of PALM
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needs a Photo-switchable probe
Imaging laser (657 nm)
Activator
Reporter
Deactivation
6000 photons
Activation
Activation laser (532 nm)
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5 µm
B-SC-1 cell, Microtubules stained with anti-ß
tubulin Cy3 / Alexa 647 secondary antibody
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5 µm
Bates et al, Science 317, 1749 1753 (2007)
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5 µm
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Standing wave illumination microscopy (SWIM)
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Also beats the Abbe limit
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Total internal reflection (fluorescence)
microscopy TIRM
z
Evanescent light wave I0, decays exponentially
with distance z from surface I0 exp(-az)
Fluorescence intensity I(z) I0
z(t) - ln I(t), fluctuating position
Distribution p(z) exp (-F(z)/kT)
Typical potential energy curves of a negatively
charged polystyrene sphere(R5 µm) close to an
equally charged glass surface as a function of
the separation distance between the glass and the
particle surface. For large particle-surface
separations the interaction potential is
dominated by gravity which can be seen in the
linear behavior of the potential curve in this
regime, whereas at small separations the
repulsive Coulomb interaction dominates. The
potential curves are plotted for particles with
different weights
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Optical near field microscope (SNOM)
D lt l
near field
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Recap

• Microscopy is all about resolution AND contrast.
• Birefringence gives information on molecular
  properties.
• Fluorescence lifetime and transfer can be used as
  contrast agents.
• Contrast (and resolution) enhancement can be
  obtained by two-photon excitation and single
  plane illumination.
• Resolution increase is possible by using
  structured illumination and ingeneous
  fluorescence excitation.