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.
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
Polarization I
property of light waves, describes orientation of
oscillations
http://www.ifsc.usp.br/~lavfis/BancoApostilasImagens/ApEfFotoelastico/photoelasticity.pdf
Polarization microscopy
Polarization II
http://www.ifsc.usp.br/~lavfis/BancoApostilasImagens/ApEfFotoelastico/photoelasticity.pdf
Birefringence
ellipticity f(d) = (n|| – n ) d/l
Results in conoscopic figures
Problem!
cholesteric liquid crystals
nematic liquid crystals
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
Birefringence image is INDEPENDENT of orientation
of optical axis
Microtubule aster image
White: 0.3nm
retardance
movie
Fluorescence
Fluorescence principle
Vibrational states
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.
Natural proteins for fluorescence studies
Green fluorescent protein (GFP)
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).
Measuring viscosities using FLIM
Measuring temperatures using FLIM
Forster resonance energy transfer (FRET)
Overlap of emission and absorption spectra of flouorophores
can be used to obtain information on their distance
FRET measures distance changes in the nanometer scale, a
relevant length scale for many biomolecules.
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.
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
Two photon fluorescence microscopy
Principle of 2 photon
fluorescence
Radiationless decay
fluoro
weakest absorption
Best penetration near 1000nm
2 NIR photons
are absorbed
simultaneously
Typical two photon fluorescence setup
High intensity required !
2 hv excit
Fluorescence detection
Selective plane illumination microscopy (SPIM)
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.
Movie of Zebrafish embryo nuclei
Stimulated emission depletion (STED)
microscopy
Typical setup
Photo-activated localization
microscopy (PALM)
The principle of PALM
The principle of PALM
needs a Photo-switchable probe
Imaging laser (657 nm)
Activator
Cy3
Reporter
Cy5
Activation laser pulses
Cy5 fluorescence
0
6000 photons
Activation
Cy3
Cy5
Cy3
5
10
Time (s)
Cy5
Activation laser (532 nm)
15
20
5 μm
B-SC-1 cell,
Microtubules stained with anti-β tubulin
Cy3 / Alexa 647 secondary antibody
5 μm
Bates et al, Science 317, 1749 – 1753 (2007)
500 nm
5 μm
Standing wave illumination microscopy (SWIM)
Also beats the Abbe limit
Total internal reflection (fluorescence)
microscopy TIRM
z
Fluorescence
intensity I(z) ~ I0
Evanescent light wave
I0, decays exponentially
with distance z from
surface I0 ~ exp(-az)
z(t) ~ - ln I(t), fluctuating position
Distribution p(z) ~ exp (-F(z)/kT)
Typical potential energy curves of a negatively charged polystyrene
sphere(R=5 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
Optical near field microscope (SNOM)
D<l
“near field “
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.