Marker-Less Stage Drift Correction in Super-Resolution Microscopy Using the Single-Cluster PHD Filter

Fluorescence microscopy is a technique which allows the imaging of cellular and intracellular dynamics through the activation of fluorescent molecules attached to them. It is a very important technique because it can be used to analyze the behavior of intracellular processes in vivo in contrast to methods like electron microscopy. There are several challenges related to the extraction of meaningful information from images acquired from optical microscopes due to the low contrast between objects and background and the fact that point-like objects are observed as blurred spots due to the diffraction limit of the optical system. Another consideration is that for the study of intracellular dynamics, multiple particles must be tracked at the same time, which is a challenging task due to problems such as the presence of false positives and missed detections in the acquired data. Additionally, the objective of the microscope is not completely static with respect to the cover slip due to mechanical vibrations or thermal expansions which introduces bias in the measurements. In this paper, a Bayesian approach is used to simultaneously track the locations of objects with different motion behaviors and the stage drift using image data obtained from fluorescence microscopy experiments. Namely, detections are extracted from the acquired frames using image processing techniques, and then these detections are used to accurately estimate the particle positions and simultaneously correct the drift introduced by the motion of the sample stage. A single cluster Probability Hypothesis Density (PHD) filter with object classification is used for the estimation of the multiple target state assuming different motion behaviors. The detection and tracking methods are tested and their performance is evaluated on both simulated and real data

Advanced single molecule localization microscopy for imaging cellular nuclei

In this PhD research, single molecule localization microscopy (SMLM) was used to image nuclear structures with a resolution down to several nanometers.The scope of this PhD research is to develop a 3D SMLM microscope which can overcome several principle limitations in imaging nuclei in 3D. The advanced improvements during this PhD research include a broad range of research subjects associated to SMLM techniques. Firstly, one of the most common problems of a super-resolution microscope is sample drift, because a small sample drift may result in artefacts and can hamper the resolution. A speckle-based method was developed to correct sample drift without changing the standard design of the SMLM setup. This drift correction method can achieve a resolution of several nanometers. Secondly, another principle problem is that commonly used organic fluorophores are restricted in their photon budget. It is often observed that the chemical structure of fluorophores change after high laser irradiance resulting in photobleaching. A patterned illumination technique was developed which allows the user to define arbitrary regions of interest for illumination with a flat-top intensity profile. Thirdly, for SMLM in particular, a carefully adjusted chemical environment in the sample is recommended to induce sufficiently blinking signals of the organic fluorophores in combination with an appropriate laser irradiance. However, such an imaging buffer can degrade over time and may not be suitable for long time imaging. Nanographene was presented as a new class of fluorophores which have blinking properties without an imaging buffer. Therefore, the nanographenes facilitate a wide range of SMLM applications including bio-imaging and material science. These advanced developments are not only for imaging nuclei, but also applicable to applications in other biological researches and in material science

Statistical Models for Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has revolutionized the field of cell biology. It allowed scientists to break the Abbe diffraction limit for fluorescence microscopy and got it closer to the electron microscopy resolution but still it faced some serious challenges. Two of the most important of these are the sample drift and the measurement noise problems that result in lower resolution images. Both of these problems are generally unavoidable where the sample drift is a natural mechanical phenomenon that occurs during the long time of image acquisition required for SMLM (Geisler et al. 2012) while the measurement noise, which arises from combining localization uncertainty and counting error, is related to the number of photons collected from the fluorophore and affects the precision in locating the centroids of single molecules (Thompson, Larson, and Webb 2002). Previous work has tried to devise methods to deal with the sample drift problem but unfortunately, these methods either add too much complexity to the experimental setup or are just inefficient in correctly estimating the drift at the single frame level (Wang et al. 2014). As for measurement noise, all current regular image rendering algorithms treat every detection of the fluorophore as a separate event and hence, the localization uncertainty of every detection of the same molecule would give offset coordinates from the other detections leading to a distorted final image. In this thesis, I demonstrate two novel approaches based on statistical concepts to address each of these two problems. The algorithm for solving the sample drift problem is based on Bayesian inference and it showed efficiency in estimating drift at the single-frame level and proved superior and more straightforward than the available methods. The algorithm for addressing the measurement noise problem is based on Gibbs sampling and not only did it enhance resolution, but it also offers for the first time a means to quantify resolution uncertainty as well as uncertainty in cluster size measurement for clustering proteins. Therefore, this work offers a significant advancement in the field of SMLM and more generally, cell biology

Quantification of Interactions between Influenza Hemagglutinin and Host Cell Phosphoinositides by Super-Resolution Microscopy

The influenza viral membrane protein hemagglutinin (HA) forms dense nanoscale clusters on host cell plasma membranes (PM), but the mechanisms that direct HA clustering are not well understood. Previous studies have observed HA associated with actin rich regions of the PM, but there are no known direct interactions between HA and actin. Phosphatidylinositol 4,5-biphosphate (PIP2) is a signaling lipid in the PM which can regulate the actin cytoskeleton, and actin comets initiated by PIP2 are known to be exploited by HA to reach the PM of infected cells. PIP2 is also used by other viruses, such as HIV and Ebola, to form clusters of viral proteins on the PM. Using diffraction-limited and super-resolution FPALM methods, we observed that HA and PH domain, a protein marker for PIP2, are closely spatially related at the PM. Clusters of PIP2 are also significantly altered in both density and area in the presence of high levels of HA, while HA clusters are significantly altered in the presence of high levels of PIP2, suggestive of an interaction between the two. Although HA mutates rapidly, there are 3 cysteines and 1-2 basic residues in the cytoplasmic tail domain (CTD) which remain highly conserved among HA subtypes. These cysteines are known to undergo palmitoylation in the Golgi, a post-translational modification where hydrophobic palmitic acids are attached. Using HA mutants and super-resolution FPALM, we examined the role of both palmitoylation and charge on the clustering properties of HA and spatial association with PIP2. Mutation of the cysteines or basic residues causes significant reductions to cluster densities (relative to cell average), while mutation of the charges appears to modulate association with PIP2. The greatest changes were observed when both the cysteines and net charge of the HA CTD were changed, causing a maximal 22% ± 6% reduction in the radial distribution functions (RDF) of clusters and a maximal 30% ± 15% increase in associated PH domain RDF amplitude. Cluster properties, density, perimeter, and circularity were also significantly affected. Even though clusters were not eliminated through CTD mutations, these findings suggest that the CTD of HA does play a role in the clustering of HA and spatial association with PIP2

Super-resolution mapping of glutamate receptors in <i>C. elegans</i> by confocal correlated PALM

Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals

Development of new approaches for characterising DNA origami-based nanostructures with atomic force microscopy and super-resolution microscopy

DNA nanotechnology has developed a versatile set of methods to utilise DNA self-assembly for the bottom-up construction of arbitrary two- and three-dimensional DNA objects in the nanometre size range, and to functionalise the structures with unprecedented site-specificity with nanoscale objects such as metallic and semiconductor nanoparticles, proteins, fluorescent dyes, or synthetic polymers. The advances in structure assembly have resulted in the application of functional DNA-based nanostructures in a gamut of fields from nanoelectronic circuitry, nanophotonics, sensing, drug delivery, to the use as host structure or calibration standard for different types of microscopy. However, the analytical means for characterising DNA-based nanostructures drag behind these advances. Open questions remain, amongst others in quantitative single-structure evaluation. While techniques such as atomic force microscopy (AFM) or transmission electron microscopy (TEM) offer feature resolution in the range of few nanometres, the number of evaluated structures is often limited by the time-consuming manual data analysis. This thesis has introduced two new approaches to quantitative structure evaluation using AFM and super-resolution fluorescence microscopy (SRM). To obtain quantitative data, semi-automated computational image analysis routines were tailored in both approaches. AFM was used to quantify the attachment yield and placement accuracy of poly(3-tri(ethylene glycol)thiophene)-b-oligodeoxynucleotide diblock copolymers on a rectangular DNA origami. This work has also introduced the first hybrid of DNA origami and a conjugated polymer that uses a highly defined polythiophene derivative synthesised via state-of-the-art Kumada catalyst-transfer polycondensation. Among the AFM-based studies on polymer-origami-hybrids, this was the first to attempt near-single molecule resolution, and the first to introduce computational image analysis. Using the FindFoci tool of the software ImageJ revealed attachment yields per handle between 26 - 33%, and determined a single block copolymer position with a precision of 80 - 90%. The analysis has pointed out parameters that potentially influence the attachment yield such as the handle density and already attached objects. Furthermore, it has suggested interactions between the attached polymer molecules. The multicolour SRM approach used the principles of single-molecule high-resolution co-localisation (SHREC) to evaluate the structural integrity and the deposition side of the DNA origami frame “tPad” based on target distances and angles in a chiral fluorophore pattern the tPads were labelled with. The computatinal routine that was developed for image analysis utilised clustering to identify the patterns in a sample’s signals and to determine their characteristic distances and angles for hundreds of tPads simultaneously. The method excluded noise robustly, and depicted the moderate proportion of intact tPads in the samples correctly. With a registration error in the range of 10 -15 nm after mapping of the colour channels, the precision of a single distance measurements on the origami appeared in the range of 20 - 30 nm. By broadening the scope of computational AFM image analysis and taking on a new SRM approach for structure analysis, this work has presented working approaches towards new tools for quantitative analysis in DNA nanotechnology. Furthermore, the work has presented a new approach to constructing hybrid structures from DNA origami and conjugated polymers, which will open up new possibilities in the construction of nanoelectronic and nanophotonic structures

Super-resolution Microscopy: Novel Developments and Optimisations

This thesis describes the design, development and optimisation of a multifunctional localisation based super-resolution microscope at Cambridge Advanced Imaging Centre. The microscope is optimised to perform single and dual-colour imaging with high localisation precision and accuracy. Moreover, three-dimensional imaging capability is included in the microscope using the double-helix point spread function and the light field imaging modality. The thesis also describes the application of the microscope to image challenging biological samples, in collaboration with the research groups at the Department of Physiology, Development and Neuroscience. This includes, studying dynamics of a DNA-binding transcription factor for the Notch signalling pathway, deep within the whole salivary glands of Drosophila. Characterisation and optimisation of the microscope and the subsequent image analysis pipeline, to extract dynamics of single molecules at such depths is also discussed. Another application of the microscope, discussed in the thesis, is the study of chromatin architecture in primary spermatocytes of Drosophila. This includes optimisation of imaging conditions and data analysis software to reconstruct features with different densities of labelling dye in the imaged nuclei. Calibration and application of dual-colour localisation microscopy, to visualise the arrangement of active transcription sites in chromatin fibres is also discussed. Finally, the thesis also presents the application of light field imaging technique to extend the depth of field of localisation microscopy to over 20 μm. Modification of the microscope for light field imaging and a method to localise point emitters with high precision in all three spatial dimensions is discussed. The effectiveness of the technique for single molecule imaging is shown by detecting emissions from single fluorophores in labelled cells

Investigating the kinetochore complex in Schizosaccharomyces pombe using advanced fluorescence microscopy techniques

Major insights into various biological processes and structures could be achieved using fluorescence microscopy, which is a non-invasive, live- and fixed cell compatible, high contrast imaging technique. Here, the molecule of interest can be specifically labeled with a fluorescent marker using a multitude of different labeling techniques, best suited for the individual biological research question. The location of the molecule of interest can be deduced from the emitted fluorescent signal of the marker due to their immediate proximity. However, structures smaller than the diffraction limit of light of about 200 nm can not be resolved by conventional fluorescence microscopy. Other techniques, such as e.g. electron microscopy (EM) or X-ray crystallography allow for higher resolutions, but lack the target specific read-out or are not compatible with in vivo studies. Nevertheless, by utilizing advanced optical components and illumination patterns and designing fluorophores with tightly controllable photophysics, the diffraction limit of light could be circumvented leading to improved resolutions. From these super-resolution techniques, only single-molecule localization microscopy (SMLM) allows for a quantitative analysis of the target molecule due to the spatiotemporal detection of the fluorescent marker. The segregation of sister-chromatids to the corresponding daughter cells is a vital and irreversible process, which needs to be tightly regulated. Here, a multi-protein complex called the kinetochore (KT), which serves as a force-sensing linker between the centromere in chromosomes and kinetochore microtubules (kMTs) originating from the spindle pole body (SPB), plays a pivotal role as errors in this process lead to aneuploidy or cell death. Thus, understanding the architecture and regulation of this complex is essential. However, even though certain subcomplexes of the KT could be resolved by EM or X-ray crystallography in vitro, the full KT nanostructure was not resolved in vivo yet. Hence in chapter 2, the in vivo nanoscale structure of the fission yeast KT complex was investigated using SMLM. The fission yeast Schizosaccharomyces pombe was used as the model organism of choice, due to its small regional centromeres. It acts as an intermediate between the point centromere in the budding yeast Saccharomyces cerevisiae, on which only one KT assembles, and the larger regional centromeres in humans. To investigate the KT in fission yeast, a structure smaller than the diffraction limit of light, different SMLM imaging and labeling strategies in microbes were developed or applied fitting this research question. However, for the creation of the KT map at least two different super-resolved targets are required: one reference protein at the centromere and one protein of interest (POI) a time in the KT complex. As no combination of commonly used photoswitchable organic dyes for SMLM proved to be applicable in fission yeast, the focus was shifted towards photoactivatable and - convertable fluorescent proteins (FPs) as alternative fluorescent markers. Knowing this, the KT structure was investigated using a multi-color SMLM approach based on FPs utilizing an orthogonal sequential illumination pattern and a KT protein database was generated. Developing novel image analysis tools and controls allowed for the extraction of intra- KT distances and POI copy numbers. Based on these parameters, first conclusions on the structure, preferred KT assembly pathways and stoichiometries were drawn and a model of the fission yeast KT was proposed. Finally, to investigate the KT structure in even greater detail, a new imaging technique combining expansion microscopy (ExM) and SMLM, termed single-molecule expansion microscopy in fission yeast (SExY) was developed in chapter 3, which increases the imaging resolution of SMLM by the corresponding expansion factor (EF) of the sample. For this, the fixed sample was first embedded in a hydrogel and then expanded upon incubation in aqueous media. To achieve an even expansion, the proteins were covalently linked to the gel mesh and obstacles like protein connections, cell walls or membranes were dissolved in homogenization steps prior to expansion. Then, the sample was imaged using the SMLM based imaging technique photoactivated localization microscopy (PALM), which lead to single-digit nanometer resolutions. Since KT proteins are low abundant, we optimized for an increase in protein retention yield, which we could improve by half compared to the initial protocol. We also optimized for an isotropic expansion of the sample, which we controlled by determining the EFs of different cell organelles and the distribution of cytosolic FPs compared to non-expanded cells. With the final SExY protocol at hand we were than able to visualize KT proteins as well as other nuclear targets in vivo at a single digit nanometer range for the first time