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
All life on earth contains DNA, which is used to store biological information. Organisms compact their DNA in order for it to fit inside their cell(s). Specialized proteins, which are referred to... Show moreAll life on earth contains DNA, which is used to store biological information. Organisms compact their DNA in order for it to fit inside their cell(s). Specialized proteins, which are referred to as nucleoid-associated proteins (NAPs), help organize the DNA to keep it compact but accessible for transcription. These NAPs often function as DNA benders, bridgers and wrappers. In most eukaryotic and archaeal species, histones are important NAPs that are able to bend or wrap the DNA duplex around its core. In this thesis, the histones from archaea are described in terms of their interaction with DNA. We describe archaeal histones on a molecular level and analyze their primary structure. We also characterize recombinant expressed archaeal histones using single molecule techniques such as tethered particle motion and magnetic tweezers. Experiments using these techniques show that some archaeal histones form a rod-shaped structure together with DNA in solution. This hypernucleosome strongly compacts DNA. Furthermore, we discuss the impact of hypernucleosome formation on transcription regulation in archaea. 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
Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation... Show moreUnderstanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction. (C) 2015 Published by Elsevier B.V. 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
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing 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
Al voordat het grote publiek kennismaakte met nanotechnologie, besteedde de wetenschap aandacht aan steeds kleinere objecten. Sinds 15 jaar is het bijvoorbeeld mogelijk om met speciale microscopen... Show moreAl voordat het grote publiek kennismaakte met nanotechnologie, besteedde de wetenschap aandacht aan steeds kleinere objecten. Sinds 15 jaar is het bijvoorbeeld mogelijk om met speciale microscopen naar individuele moleculen te kijken! Deze techniek hebben we nu gebruikt om halfgeleider nanokristallen te bestuderen. Halfgeleiders zijn materialen die worden gebruikt in computers. Afhankelijk van hun bewerking en toepassing kunnen zij stroom goed of slecht geleiden. Maar deze nanokristallen zijn zo klein, slechts 5 nanometer (1 miljoen nm = 1 mm) en dus veel kleiner dan de golflengte van licht, dat ze zich heel anders gedragen. Ze kunnen bijvoorbeeld licht absorberen en uitzenden (fluoresceren), maar omdat ze zo klein zijn altijd slechts __n lichtdeeltje (foton) tegelijk, net als een molecuul. Ook het ritme (de statistiek) waarmee dit gebeurt, is heel bijzonder en vergelijkbaar met de statistiek waarmee aardbevingen voorkomen. De pauze tussen het uitzenden van twee fotonen varieert bijvoorbeeld zo sterk, dat we de gemiddelde pauze niet kunnen uitrekenen! Dankzij diverse wiskundige trucs kunnen we dit ritme nu toch beschrijven. Deze wiskunde is ook handig om verschillende experimenten te kunnen vergelijken, zowel voor nanokristallen als voor moleculen. Vervolgens hebben we een model opgesteld dat beschrijft wat er in nanokristallen gebeurt, als een foton wordt geabsorbeerd of uitgezonden. Show less
