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
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
