By Prof. Jörg
Single molecule spectroscopy and imaging
Fluorescence spectroscopy and microscopy is one of the most advanced and
important techniques in biophysical research. The enormous sensitivity of
fluorescence allows for the direct detection, spectroscopy and imaging of
individual molecules. During the last 20 years, this has led to completely new
insights into the functioning, dynamics and interaction of individual
bio-molecules such as proteins, DNA or RNA. Moreover, single molecule
spectroscopy offers a unique way to directly observe the stochastic nature of
molecular processes and thus connects advanced biophysics with fundamental
statistical physics. Furthermore, due to the non-invasive character of
fluorescence detection it is possible to track and watch individual molecules
inside living cells. Last but not least, fluorescence microscopy itself has seen
a dramatic development over the last decade, improving its resolving power by
more than one order of magnitude, allowing for resolving cellular structures of
only a few dozen nanometers wide.
Single-molecule fluorescence spectroscopy
Fluorescence correlation spectroscopy is a powerful technique to study
molecular dynamics and interaction. In fluorescence correlation spectroscopy,
fast fluctuations of fluorescence intensity generated by single molecules within
a very small volume of only one picoliter (10-15 liter) are recorded
and analyzed by correlation. One of the most important applications of this
technique is to study protein folding. Proteins are the most important molecular
building blocks of life, consisting of highly ordered structure made of long
chains of amino acids. The mechanisms how a disordered long chain of amino acids
(unfolded protein) finds the highly ordered final structure of the folded
protein within a short time has remained one of the big questions of biophysics
since the emergence of this problem some 50 years ago. The ability to watch the
folding (and unfolding) of individual proteins offers an unprecedented view on
this process and yields invaluable information for a better theoretical
understanding of the protein folding paradox.
|Schematic of a single-molecule fluorescence spectrometer:
Three excitation lasers with different wavelengths are combined and that light
is reflected by a dichroic mirror (reflective for laser excitation, transparent
for fluorescence emission) into the objective, which focuses it into a tiny
spot. Excited fluorescence light is collected by the same objective, focused
through a confocal pinhole for background suppression, and finally detected by
single-photon sensitive detectors.|
|Transition between unfolded (left) and folded (right) state
of a mini-protein: A core application of single-molecule fluorescence
spectroscopy is to study the temporal dynamics and pathways of the fast
transitions between these states.|
Single-molecule fluorescence imaging
Besides watching the fluorescence fluctuations of a single molecule, one can
nowadays directly image single molecules using specialized high-sensitivity CCD
cameras. This is the core technique when using single molecule fluorescence for
following molecular processes in living cells. The ability to ‘see’ a single
molecule allows for localizing its position in space with nanometer accuracy,
far below the conventional resolution-limit of a light microscope. This has been
used in the past to resolve cellular structures with nanometer accuracy, or to
watch the motion of individual proteins with similar resolution. We have
developed a particular version of single-molecule imaging which enables us not
only to see its position in space but also its three-dimensional orientation. In
combination with the positional information, we are thus able to watch the
complete motion and rotation of individual molecules and to elucidate how a
protein turns and moves when functioning and interacting with other molecules.
The same method is also used to study the local structure and dynamics of
polymers and complex liquids.
|Angular distribution of emission of a single molecule
(inset) together with observed defocused images of single molecules: Each
observed “double-banana” shaped pattern stems from a single molecule and
contains information about its three-dimensional orientation in
Super-resolution fluorescence microscopy
Fluorescence microscopy is one of the most important tools when studying the
architecture and structure of cells and tissues. The main reasons for this are
its exceptional sensitivity (even individual molecules can be ‘seen’), its
non-invasiveness (using moderate light intensities does not harm a cell, in
contrast to electron microscopy, that can only be performed on dead samples),
and its specificity (the possibility to label different molecules of interest
with different and well distinguishable fluorescent dyes). Unfortunately, for
many years the spatial resolution of fluorescence microscopy was limited to ca.
250 nm which is due to the wave nature of light. Electron microscopy achieves a
spatial resolution of up to three orders of magnitude better, using the short
quantum wavelength of energetic electrons, but can be used only on treated dead
samples. In recent years, the classical resolution limit of fluorescence
microscopy has been overcome by exploiting various non-linear properties of
fluorescence excitation and detection. Our group has developed two promising and
rapidly evolving techniques of super-resolution fluorescence microscopy:
Superresolution Fluctuation Imaging Microscopy (SOFI) and Image Scanning
Microscopy (ISM), which allow for three-dimensional and multicolor
superresolution imaging of live and fixed cells and tissue.
|Left panel: Conventional wide-field image of dividing HEK
cells where the tubulin network is labeled with fluorescent quantum dots. Right
panel: Same cells in SOFI resolution – the tubulin network is visible in much
higher resolution, contrast and finer
From an electrodynamic point of view, fluorescent molecules can be understood
as nanoscopic antennas that absorb and emit electromagnetic radiation. These
nano-antennas probe the local density of states of the electromagnetic field. By
placing them in nanometric metallic and/or dielectric structures, one can study
how these structures alter the local density of states by measuring the changes
in absorption and emission of the molecular fluorescence. This is of enormous
importance for our fundamental understanding of the interaction between light
and matter, but offers also a fascinating way to tune and tailor the
fluorescence properties of molecules. Our group studies fluorescence nano-optics
with sophisticated numerical modeling, and by performing advanced
single-molecule spectroscopy measurements.
|Angular distribution of emission of a single molecule placed
within a micro-cylinder. Shown are the distributions for two different molecule
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