DNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin....Show moreDNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin. Little is known of the effect of this chromatin structure on the conformational dynamics of nucleosomes. Here we introduce a single-pair F¨orster Resonance Energy Transfer (spFRET) method that allows for quantitative measurement of nucleosome structure in folded fibers through both Fluorescence Correlation Spectroscopy (FCS) and burst analysis. Preliminary experiments determined optimal measurement concentrations and methods of excitation. However, measurements on reconstituted chromatin fibers showed poor signal-to-noise. We propose several improvements to enable the study of chromatin dynamics, such as nucleosome breathing. We expect the work outlined in this thesis to contribute to greater understanding of both nucleosome and chromatin structure, and how these regulate the accessibility of DNA to other molecules and proteins.Show less
Force spectroscopy on single molecules is a technique that is used to study the mechanical and structural properties of biomolecules. A relatively new force spectroscopy technique is acoustic force...Show moreForce spectroscopy on single molecules is a technique that is used to study the mechanical and structural properties of biomolecules. A relatively new force spectroscopy technique is acoustic force spectroscopy (AFS), which exploits acoustic waves to generate a wide range of forces with high pulling rates. A piezoelectric transducer is used to generate acoustic waves at specific resonance frequencies. Acoustic standing waves exert forces on micro-particles and pull them to acoustic pressure nodes. However, commercially available AFS flow cells are expensive and users find them nontrivial to operate. Here we present a new AFS flow cell design where we placed the piezoelectric transducer directly on top of the fluid channel of the flow cell to reduce the loss of acoustic pressure between the transducer and fluid. We used 3D finite element method simulations and found that the acoustic pressure profile is more complex than expected due to acoustic variations in the xy-plane. However, we found that the dimensions of the flow cell can be optimised to minimise acoustic variations in the xy-plane, demonstrating that more homogeneous pulling forces can be achieved. We performed AFS measurements on the new flow cell design, but we were not able to demonstrate an acoustic force. We anticipate this study helps to further optimise the AFS flow cell design, resulting in maximised pulling forces with minimal variations in the xy-plane.Show less
We introduce an open source, freely available algorithm for automatic correlation of fluorescence and electron microscopy images in Correlative Light-Electron Microscopy (CLEM). With a minimal...Show moreWe introduce an open source, freely available algorithm for automatic correlation of fluorescence and electron microscopy images in Correlative Light-Electron Microscopy (CLEM). With a minimal amount of manual input, two custom modules perform an automatic identification of the fluorescent nuclei and the watershed nucleus of a TEM image and hence calculate the necessary affine transformation for accurate correlation. The process can be performed on a group of pre-processed images and allows for an efficient solution around the data handling bottleneck encountered in the technique.Show less
In eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to...Show moreIn eukaryotic cells, nucleosomes regulate DNA processes such as transcription, replication and repair. The dynamic architecture of nucleosomes is the key for understanding gene regulation. Due to the compacted structure of nucleosome, these dynamics are a challenging to study. Fluorescence Resonance Energy Transfer (FRET) microscopy combined with a nucleosome immobilization strategy, allows to observe conformational dynamics over long time scales. However, it is not trivial to immobilize nucleosomes properly, avoiding impurities or non-targeted molecules that could perturb measuring nucleosome dynamics. Here we present an optimized sample preparation protocol for studying conformational dynamics in nucleosomes. Using an immobilization strategy based on PLL-g-PEG/PEG-biotin and NeutrAvidin, we found a limited immobilization specificity and structural integrity of nucleosomes, possibly due to the influence of the experimental conditions as the measurements settings and the surface preparation. Nevertheless, we were able to identify some nucleosome FRET dynamics using this technique. Furthermore, an innovative method for cleaning coverslips and quantifying the cleaning quality, open a window of possibilities for automated sample preparation protocols in single molecule studies.Show less
Complex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular...Show moreComplex cellular processes can be characterised using single particle tracking techniques (SPT). ‘Labels’ such as metal nanoparticles are introduced into cells and tracked to reveal molecular trajectories. Many current techniques are based on fluorescence microscopy, and have nanometre-resolution. To accurately probe cellular processes, a technique must also have long-term 3D in-vivo observing capability with a minimal toxic effect. Gold nanorods (GNRs) in two-photon microscopy is a promising technique. GNRs are non-toxic, easily functionalisable, and exhibit a bright two-photon fluorescence. However, the theoretical positional accuracy for this technique is not yet known. Furthermore, the detailed trajectory data present statistical challenges. We have addressed these issues here numerically, using simulated image data. We found the accuracy to be between 5.2nm and 8.9nm for stationary GNRS, dependent on the separation between slices. We also found that diffusive movement of a nanorod lowers the accuracy, at worst case to 31.8nm. We have also investigated optimising the extraction of behaviour properties from MSD plots, and have used Welsh’s test to detect transitions. GNRs in two-photon microscopy has been shown to be a very accurate technique, and its trajectory data can yield accurate behavioural information. The fit of the PSF may be improved, but the techniques already compares well against others.Show less
In eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it...Show moreIn eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it accessibility for DNA binding proteins. Conformational dynamics of the nucleosome, like partial release of the DNA, called nucleosome breathing, plays an important part in regulating this accessibility of the genetic information. To study the wrapping and unwrapping in DNA breathing we followed the Förster Resonance Energy Transfer of a pair of fluorophores placed in the nucleosome, in time. We selected a single nucleosome immobilized on glass from an image acquired by a scanning confocal microscopy. Data acquisition and analysis software was developed to record and process time trace of individual nucleosomes with sub ms resolution. Although we can now reach the required temporal resolution to resolve nucleosome breathing, we did not observe it. A large fraction of the nucleosomes did not show FRET after surface immobilization suggesting partial disassembly, which prevented statistical analysis of large numbers of nucleosomes.Show less
