Implementing super-resolution palm microscopy in fission yeast

Fluorescence microscopy is a popular biological technique because it allows the study of cells in great detail. However, the resolution achievable is limited by the diffraction properties of light, meaning that fine detail cannot be resolved. Various super-resolution microscopy methods have been developed to break this resolution limit. This thesis focuses on the single molecule localisation microscopy techniques. My host laboratory focuses on DNA replication and repair pathways using the model organism Schizosaccharomyces pombe (fission yeast). The aim of this thesis is thus to apply the technique of photo-activatable localisation microscopy (PALM) to specific biological questions in order to establish its benefits and limitations. In theory, in PALM every molecule will be imaged once and, as such, could be counted. So far this has been largely limited to membrane proteins. Using a combination of artificially created fluorescent oligomers, endogenous ribonucleotide reductase proteins tagged with mEos and computer simulations I studied the feasibility of counting highly expressed cytoplasmic proteins and assigning them to complexes of known or unknown stoichiometry. I established that density of expression is a significant limiting factor when using PALM to resolve complex stoichiometry. I thus went on to develop a variation of fluorescence correlation spectrometry to study the same protein complexes to see if we could determine their stoichiometry by diffusion speed. I established that the technique could differentiate between quite small changes in size. However the endogenous complex did not respond well to the fluorophore used so I was not able to establish its size. Using the PALM system I also studied a biological molecule, Rrp2, which was expressed at such low levels it was not possible to observe with conventional fluorescence microscopy. I established that we were able to observe this protein at endogenous levels and characterised its behaviour in response to stress

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.We thank the Wellcome Trust for the PhD studentship of MP (093756/B/10/Z), and the Royal Society for the University Research Fellowship of SFL (UF120277). The work by SB and DL was also funded by the Wellcome Trust (082010/Z/07/Z). UE and MH acknowledge funding by the German Science Foundation (grants EXC 115 and SFB 902). SB is funded by a BBSRC grant (BB/K013726/1). AMC acknowledges ERC Award 268788-SMI-DDR. We also thank the European Commision for support through the 4DCellFate project (EC FP7 CP 277899).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.012543

Polymersome-Mediated Delivery of Combination Anticancer Therapy to Head and Neck Cancer Cells: 2D and 3D in Vitro Evaluation

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors

Proceedings of Patient Reported Outcome Measure’s (PROMs) Conference Oxford 2017: Advances in Patient Reported Outcomes Research

A33-Effects of Out-of-Pocket (OOP) Payments and Financial Distress on Quality of Life (QoL) of People with Parkinson’s (PwP) and their Carer

Use of UK faith Centre as a COVID-19 community vaccination clinic: exploring a potential model for community-based healthcare delivery

Introduction: Effective and safe vaccinesagainstCOVID-19areessentialtoachieveglobalcontrolofthecoronavirus(SARS-CoV-2).Using faith centres may offer a promising route for promoting higher vaccine uptake from certain minority ethnic groups known to be more likely to be vaccine hesitant. Methods: This cross-sectional study explored attendees’ perceptions, experiences of being offered, and receiving COVID-19 vaccination in a local mosque in Woking, Surrey, UK. About 199 attendees completed a brief questionnaire on experiences, views, motivations about attending the mosque and vaccination on site. Results: The most common ethnic groups reported were White British (39.2%) and Pakistani (22.6%); 36.2% identified as Christian, 23.6% as Muslim, 5.5% as Hindu, and 17.1% had no religion. Genders distribution was relatively equal with 90 men (45.2%) and 98 women (49.2%), and 35–44-year-old represented the most common age group (28.1%). Views and experiences around receiving vaccinations at the mosque were predominantly positive. Primary reasons for getting vaccinated at the mosque included convenience, accessibility, positive aspects of the venue’s intercultural relations, and intentions to protect oneself against COVID-19, regardless of venue type. Negative views and experiences in regards to receiving the vaccination at the mosque were less common (7% expressed no intention of recommending the centre to others), and disliked aspects mostly referred to the travel distance and long waiting times. Conclusions: Offering COVID-19 vaccination in faith centres appears acceptable for different faith groups, ensuring convenient access for communities from all religions and ethnic backgrounds

Structural imaging mode.

<p>Fixed <i>S</i>. <i>pombe</i> expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting <b>(A)</b>, or after applying the vlsPALM thresholds defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125438#pone.0125438.g002" target="_blank">Fig 2B</a><b>(B)</b>. The corresponding diffraction-limited image of the two cells is shown as an inset in <b>(A)</b>. Close-ups of the white rectangles in <b>(A-B)</b> are shown in <b>(C-D)</b>. The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in <b>(C-D)</b>).</p

Confidence mode.

<p>Embryonic stem cells expressing Cenp-A-mEos proteins were fixed and imaged. The corresponding movie (summed in <b>A</b>) was analysed with Peak Fit and the resulting list of localisations was separated between in vls (green) and out of vls (red) localisations using the vlsPALM thresholds defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125438#pone.0125438.g002" target="_blank">Fig 2C</a>. vlsPALM allows the identification of the in-focus localisations <b>(B)</b>. All localisations were plotted either as fitted <b>(C)</b> or in a super-resolved picture <b>(D)</b>, but coloured according to the vlsPALM filtering. Three categories of Cenp-A clusters were observed: some were almost entirely within the vls (<b>D-F</b>, 1); others were spanning one extremity of the vls, partly in the vls (<b>D-F</b>, 2); the last ones were entirely out of the vls (<b>D-F</b>, 3). <b>(E)</b> shows the diffraction-limited and super-resolved close-ups of the Cenp-A clusters defined in <b>(D)</b>. <b>(F)</b> displays the number of localisations in (green) and out of (red) the vls for each cluster. Such classification allows selecting in-focus clusters for further quantification and preventing under-counting due to undetectable out-of-focus emitters.</p

Variation of the PSF in three dimensions.

<p>A vls (green plane, <b>A</b>) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, <b>A</b>). A fluorophore emitting from outside the vls (red volume above and below the vls, <b>A</b>), is blurred on the image plane of the detector (red optical path, <b>A</b>). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume <b>(B)</b> of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in <b>(C-E)</b>. For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.</p