Oscillations, Waves, and Interactions - GWDG

436 S. Lakämper and C. F. Schmidt
interest. In contrast to conventional microscopy, the AFM reports the response of the
imaged objects to the force exerted by the tip which can give more than just structural
information about the sample. While generally rather slow compared to, e. g.,
video microscopy, AFM provides nanometre or better resolution and therefore can
resolve details of bio-macromolecules that are otherwise only accessible by electron
microscopy or X-ray crystallography.
The AFM furthermore allows one to measure forces with piconewton resolution.
This capability can be used to mechanically probe single molecules, but also biopolymers,
such as DNA or protein chains, and determine rigidity, rupture forces or unfolding
forces. AFM-imaging of biomolecules is generally still a slow technique, requiring
10 s or more of seconds per frame. We are especially interested in new developments
aiming for imaging at video rates in order to capitalize on the capability of AFM to
monitor structure, mechanics and dynamics at the same time in physiological conditions.
AFM can also be combined with fluorescence microscopy which adds specific
recognition.
1.2 Fluorescence microscopy
Light microscopy and especially fluorescence microscopy has experienced a renaissance
with the advent of laser illumination and highly photo-stable chemical and biological
fluorophores. We are especially focussing on the imaging of single molecules.
The development of specific labelling strategies and of highly sensitive detection
methods and cameras have made the real-time observation of single molecules – in
vitro – or even in living organisms – in vivo – possible.
To be able to detect single molecules, the background fluorescence needs to be
sufficiently low. Two approaches are used in the lab: using total internal reflection
of a laser-beam on the glass–water interface of the sample chamber, the (evanescent
wave) illumination-depth within the sample is reduced to 100–200 nm. This
drastically reduces the background fluorescence, as the typically tens of µm-thick
samples are not completely illuminated. The other approach is to use wide-field illumination
and strongly reduce the concentration of active fluorescent proteins. The
latter approach allows better control over illumination intensities and the bleaching
processes. Furthermore, we are currently developing multi-colour single molecule fluorescence
setups to use Förster-Resonance Energy Transfer (FRET) between pairs
of fluorophores. Since the energy transfer is strongly dependent on distance, it can
be used to monitor domain and/or sub-unit interactions of proteins (molecular motors/chaperonins)
on the nanometre scale.
Diffraction-limited imaging of single fluorophores results in diffraction patterns
of size ∼ λ/2 which limits the spatial resolution in densely labeled samples. An
individual fluorophore can, however, be localized by fitting the diffraction pattern of
a point source with accuracies better than 2 nm, which provides valuable information
about the dynamics of molecular machines. The accuracy of position detection and
relative shifts can be combined with or complemented by the sub-nm resolution of
optical trapping techniques.