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 space.
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 detail.
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 orientations.
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