Detecting nanoscopic objects plays an important role in nanoscience in particular, in the rapidly growing field of nanobiology. The forebear to modern super-resolution microscopy for single... Show moreDetecting nanoscopic objects plays an important role in nanoscience in particular, in the rapidly growing field of nanobiology. The forebear to modern super-resolution microscopy for single molecule investigation, is fluorescence microscopy. Fluorescence as a contrast mechanism, however, brings several restrictions. These include (1) the use of the label itself, which may introduce artifacts to the interpretation, (2) the limited photoemission caused by photobleaching and photoblinking as well as (3) low bandwidth of the emission. Fluorescence-free alternatives are thus highly desirable to overcome these limitations. Optical detection of individual proteins with high bandwidth holds great promise for understanding important biological processes on the nanoscale. In this thesis, we investigate label-free optoplasmonic detection of single proteins and particles in motion. Analysing the data provide information about the hydrodynamic volume of the diffuser and interaction such as binding events. Show less
We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing... Show moreWe developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule fluorescence correlation spectroscopy (FCS) of the fluorescently labeled Copper azurin in solution reveals how the position of the label in the 3-D structure of the protein affects the redox kinetics of the redox center as well as the label. Under certain redox conditions, we have been able to observe a microsecond dynamics for intramolecular ET reaction between the label and the metal center in azurin. Our results show that this FRET technique can be profitably used to study the enzyme activity of dye-labeled oxidoreductases. Show less
We study the technique of photothermal microscopy by which we can detect single nano-objects by their absorption at room temperature. We optimize the sensitivity of this technique and demonstrate... Show moreWe study the technique of photothermal microscopy by which we can detect single nano-objects by their absorption at room temperature. We optimize the sensitivity of this technique and demonstrate the first optical detection of a single molecule by its absorption at room temperature. Moreover, we combine photothermal, luminescence and scattering of individual nano-objects (organic dye nanoparticles and gold nanoparticles) at single-particle level to gain insight into their radiative and nonradiative properties. Single organic nanoparticles exhibit a complex excitation power-dependent luminescence quantum yield due to singlet-singlet or singlet-triplet annihilation, and their luminescence quantum yield can be as high as 10^(__2). In contrast to organic dye nanoparticles, gold nanoparticles yield very stable optical signals. Gold nanoparticles are also easily detectable by their photoluminescence. We find that the luminescence quantum yield of single gold nanoparticles is nearly independent of their volumes and can be as high as ~10^(-5) for nanorods with a plasmon resonance of ~650 nm. We further investigate the sensitivity of a single gold nanorod to an approaching dielectric surface. We show that the nanorod exhibits significant red-shift in its plasmon resonance wavelength for distances less than 400 nm pointing the way towards the possible application of nanorods as distance sensors. Show less
Gold nanoparticles are spherical clusters of gold atoms, with diameters typically between 1 and 100 nanometers. The applications of these particles are rather diverse, from optical labels for... Show moreGold nanoparticles are spherical clusters of gold atoms, with diameters typically between 1 and 100 nanometers. The applications of these particles are rather diverse, from optical labels for biological experiments to data carrier for optical data storage. The goal of my project was to develop new methods to study the physical properties of single gold nanoparticles on ultra-short timescales. Exciation with a short laser pulse brings a nanoparticle out of equilibrium, which makes it vibrate with a period that depends on the particle diameter and the speed of sound in gold. The vibrational period of a gold nanoparticle with a diameter of 60 nanometer is 20 picoseconds. This acoustic vibration has been detected by us for the first time for single particles. The main advantage of single-particle studies over bulk detection of these particles lies in the fact that all particles in an ensemble vibrate at slightly different frequencies, which causes increased damping due to dephasing. The damping of the vibrations of single particles only depends on the elastic coupling between the particle and its environment, which offers the possibility of using these particles as mechanical nanosensors. Show less
