In this work, magnetic tweezers were used to study the locomotion of active particles in bulk. For this purpose half platinum-coated magnetic spherical Janus particles were suspended in a 5% H2O2...Show moreIn this work, magnetic tweezers were used to study the locomotion of active particles in bulk. For this purpose half platinum-coated magnetic spherical Janus particles were suspended in a 5% H2O2 solution, by which active locomotion was induced. The substrate-particle distance was varied by means of a magnetic field in a magnetic tweezers set-up. By recording the diffraction pattern and the (x,y) positions of these particles during locomotion, the velocity and translational diffusion coefficients were calculated from their Mean-Squared Displacement (MSD) and compared to near-wall locomotion. Although not enough data was gathered to draw conclusions on the influence of the wall on the locomotion of the particles, the applied method appears promising to do so in future research. Namely, it is shown that the magnetic particles can clearly be elevated and be retained in a range of height in bulk. The method is discussed extensively and suggestions for improvements or other approaches are given in the discussion.Show less
As the basic unit of chromatin, the form in which DNA is tightly packed in the nucleus of eukaryotic cells, the nucleosome forms a physical barrier during transcription of the DNA. Understanding...Show moreAs the basic unit of chromatin, the form in which DNA is tightly packed in the nucleus of eukaryotic cells, the nucleosome forms a physical barrier during transcription of the DNA. Understanding the energetic landscape of the nucleosome during transcription extends our knowledge on how the nucleosome affects gene expression. An in vitro study of the energetic landscape of native nucleosomes has never been done. To facilitate such a study, techniques need to be developed to mechanically unzip native chromatin. In this research, we developed techniques on DNA unzipping using magnetic tweezers that are needed for the localization of nucleosomes in chromatin unzipping. We investigated long-lifetime DNA tethering to improve reproducibility and experimental practicality, which is vital for tethers containing nucleosomes. Techniques of force barrier localization during DNA unzipping were developed that could be used on nucleosomes. Two-state equilibrium statistical mechanics models for DNA unzipping and overstretching were developed that are extendable to include more states. These techniques aim to facilitate experiments on native nucleosomes that shine light on their fundamental role in epigenetics.Show less
Two-photon excitation (TPE) is the principle of simultaneous absorption of two photons to excite an electron. In biological imaging, this principle is used to observe intact tissue at high image...Show moreTwo-photon excitation (TPE) is the principle of simultaneous absorption of two photons to excite an electron. In biological imaging, this principle is used to observe intact tissue at high image depth ( 1 mm) with near infrared wavelengths. Relative to one photon fluorescence, previous studies have shown enhanced photobleaching with TPE. In this thesis we use a scanning multifocal two-photon microscope (MTPM) to observe photobleaching of fluorescent Rhodamine molecules. Previous observations with MTPM have shown limited photobleaching. However, these results were obtained for large ensembles of fluorescent molecules. In this thesis we used MTPM to observe photobleaching of spots containing a small number of fluorescent molecules in a Rhodamine-DNA complex. Time traces of individual Rhodamine-DNA complexes showed limited photobleaching. Additionally, single Rhodamine molecules were observed with a high numerical aperture (NA) objective. The single Rhodamine molecules showed an enhanced photobleaching rate compared to the Rhodamine-DNA complexes, observed with a lower NA objective. The results in this thesis suggest MTPM as a viable method for in vivo observations of single molecules with TPE.Show less
Chromatin is a dense structure of DNA and histone proteins. DNA wraps in units of 147 bps around 8 histones forming the nucleosomes. Strings of nucleosomes stack into dense 30 nm fibers, but the...Show moreChromatin is a dense structure of DNA and histone proteins. DNA wraps in units of 147 bps around 8 histones forming the nucleosomes. Strings of nucleosomes stack into dense 30 nm fibers, but the structure of these fibers remains disputed. Force spectroscopy experiments show that the linker length affects the characteristic unfolding of nucleosome-arrays and suggests two alternative conformations. However, the precise influence of linker base pairs remains unclear. Here we use Monte Carlo simulations and show that DNA twist-energy largely determines whether nucleosomes stack in a 1-start or 2-start conformation. In addition, we show with Mutation Monte Carlo simulations that the sequence of the linker DNA may affect nucleosome stacking. Thus both the sequence and length of the linker DNA are important for the higher-order structure of 30 nm chromatin fibers. Furthermore, the simulations form a bridge between theory and experiment as predicted changes in chromatin folding can be verified experimentally.Show less
Gold nanorods (GNRs) are plasmonic nanoparticles of which the plasmon resonance wavelength is influenced by the near-field surroundings. Even binding of single proteins with GNRs can cause a shift...Show moreGold nanorods (GNRs) are plasmonic nanoparticles of which the plasmon resonance wavelength is influenced by the near-field surroundings. Even binding of single proteins with GNRs can cause a shift in their resonance wavelength. Previous studies have used this resonance shift to detect single binding events of proteins with GNRs. However, those results are limited in their throughput and sensitivity. Here we use a multifocal two-photon microscopy technique to measure hundreds of single GNRs simultaneously with high spectral sensitivity and signal-to-noise ratio. Using numerical simulations we determined how we can optimise the homogeneity of the illumination pattern over an area of 200 × 200 µm2 and minimise the melting of GNRs during measurements. Finally, we measured GNRs in various concentrations of fibronectin proteins and found an increase in power spectral density of the two-photon luminescence signal for fibronectin concentrations of 1.25 µg/mL to 2.5 µg/mL. Through a better understanding of the setup, we can now perform reliable spectral measurements of hundreds of individual GNRs. This should make two-photon microscopy techniques more competitive with bio-sensing experiments based on scattering of GNRs.Show less
Gold nanorods (GNRs) have unique optical properties. GNRs can be excited in the near-infrared range and their photoluminescence is bright and stable. Because of this, GNRs have a large range of...Show moreGold nanorods (GNRs) have unique optical properties. GNRs can be excited in the near-infrared range and their photoluminescence is bright and stable. Because of this, GNRs have a large range of possible applications, including use as labels or as biosensors. For these kinds of applications, it is important to be able to determine a GNR’s properties with high accuracy. Here we characterize single gold nanorods by five properties: their 3D position, plasmon resonance and orientation. The position of GNRs is determined with a sub-nanometer error in x, y and a 3 nm error in z. The surface plasmon resonance wavelength and the orientation of GNRs are determined with errors of <0.1 nm and 0.1 deg respectively. This is achieved by applying a four-dimensional fit to a stack of two-photon photoluminescence images. The methods presented in this thesis can be used to improve accuracy in the aforementioned applications of GNRs.Show less
