70 research outputs found

    Enabling and understanding nanoparticle surface binding assays with interferometric imaging

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    There is great need of robust and high throughput techniques for accurately measuring the concentration of nanoparticles in a solution. Microarray imaging techniques using widely used to quantify the binding of labeled analytes to a functionalized surface. However, most approaches require the combined output of many individual binding events to produce a measurable signal, which limits the sensitivity of such assays at low sample concentrations. Although a number of high-NA optical techniques have demonstrated the capability of imaging individual nanoparticles, these approaches have not been adopted for diagnostics due complex instrumentation and low assay throughput. Alternatively, interferometric imaging techniques based on light scattering have demonstrated the potential for single nanoparticle detection on a robust and inexpensive platform. This dissertation focuses on the development of methods and infrastructure to enable the development of diagnostic assays using the Single Particle Interferometric Imaging Sensor (SP-IRIS). SP-IRIS uses a bright-field reflectance microscope to image microarrays immobilized on a simple reflective substrate, which acts as a common-path homodyne interferometer to enhance the visibility of nanoparticles captured near its surface. This technique can be used to detect natural nanoparticles (such as viruses and exosomes) as well as molecular analytes (proteins and nucleic acid sequences) which have been tagged with metallic nanoparticle in a sandwich assay format. Although previous research efforts have demonstrated the potential for SP-IRIS assays in a variety of applications, these studies have largely been focused on demonstrating theoretical proof of concept in a laboratory setting. In contrast, the effective use of SP-IRIS as a clinical diagnostic platform will require significant functional improvements in automation of assay incubation, instrument control, and image analysis. In this dissertation, we discuss the development of instrumentation and software to support the translation of SP-IRIS from manual laboratory technique into an automated diagnostic platform. We first present a collection of mechanical solutions to enable the real-time, in-solution imaging of nanoparticles in disposable microfluidic cartridges. Next, we present image analysis techniques for the detection of nanoparticle signatures within digital images, and discuss solutions to the unique obstacles presented by the ill-defined focal properties of homodyne interferometry. Finally, we present a particle tracking algorithm for residence time analysis of nanoparticle binding in real-time datasets. Collectively, these improvements represent significant progress towards the use of SP-IRIS as a robust and automated diagnostic platform.2019-07-02T00:00:00

    Astrocytes-derived extracellular vesicles in motion at the neuron surface: Involvement of the prion protein

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    Astrocytes-derived extracellular vesicles (EVs) are key players in glia-neuron communication. However, whether EVs interact with neurons at preferential sites and how EVs reach these sites on neurons remains elusive. Using optical manipulation to study single EV-neuron dynamics, we here show that large EVs scan the neuron surface and use neuronal processes as highways to move extracellularly. Large EV motion on neurites is driven by the binding of EV to a surface receptor that slides on neuronal membrane, thanks to actin cytoskeleton rearrangements. The use of prion protein (PrP)-coated synthetic beads and PrP knock out EVs/neurons points at vesicular PrP and its receptor(s) on neurons in the control of EV motion. Surprisingly, a fraction of large EVs contains actin filaments and has an independent capacity to move in an actin-mediated way, through intermittent contacts with the plasma membrane. Our results unveil, for the first time, a dual mechanism exploited by astrocytic large EVs to passively/actively reach target sites on neurons moving on the neuron surface

    Factors involved in the oligomerisation of the cyanide dihydratase from Bacillus pumilus C1

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    The cyanide dihydratase enzyme from Bacillus pumilus C1 (CynDₚᵤₘ) is a member of the nitrilase superfamily and is known to specifically catalyse the conversion of cyanide into formic acid and ammonia. This enzyme is a good candidate for bioremediation of cyanide waste but the high alkaline pH of the cyanide waste water poses a problem in that it inactivates the wild type enzyme and therefore improvement of stability is required in order to synthesize an effective enzyme. Over the pH range of 6–8 the enzyme exists as short 18-subunit spirals which associate to form long, more stable helical fibres at pH 5.4. The reason for this pH dependent transition is not fully understood but it is hypothesized to be due to changes in the charge of histidine residues. The aim of this project is to obtain a high resolution structure of CynDₚᵤₘ, relate this to its function, and investigate the role of the histidines in oligomerisation with aid of the structure. Using Cryo-electron microscopy techniques a three dimensional reconstruction structure of purified CynDₚᵤₘ was obtained at a resolution of ~5Å. By flexibly fitting a CynDₚᵤₘ homology model into this high resolution structure we were able to identify amino acid residues involved in oligomerisation and stability as well as the role of the histidines, with aid from additional mutagenesis studies. Interactions at the C-interfacial region were shown to play the most crucial role in oligomerisation and included the His71-Asp275 and Arg67-Asp275 interactions. Mutations at His128, His184, His241 and His285 were shown to affect the oligomerisation of the enzyme by indirectly disrupting interactions at the interfacial regions. The Q86R+H305K+H308K+H323K mutations were shown to increase the stability of the CynDₚᵤₘ by introducing a stronger arginine-arginine interaction at the D interfacial region and a new strong interaction at the C-terminal region

    Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

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    At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction

    Architecture of the RNA polymerase II-Paf1C-TFIIS transcription elongation complex

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    Multi-functional Fluorescence Microscopy via PSF Engineering for High-throughput Super-resolution Imaging

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    Image-based single cell analysis is essential to study gene expression levels and subcellular functions with preserving the native spatial locations of biomolecules. However, its low throughput has prevented its wide use to fundamental biology and biomedical applications which require large cellular populations in a rapid and efficient fashion. Here, we report a 2.5D microcopy (2.5DM) that significantly improves the image acquisition rate while maintaining high-resolution and single molecule sensitivity. Unlike serial z-scanning in conventional approaches, volumetric information is simultaneously projected onto a 2D image plane in a single shot by engineering the fluorescence light using a novel phase pattern. The imaging depth can be flexibly adjusted and multiple fluorescent markers can be readily visualized. We further enhance the transmission efficiency of 2.5DM by ~2-fold via configuring the spatial light modulator used for the phase modulation in a polarization-insensitive manner. Our approach provides a uniform focal response within a specific imaging depth, allowing to perform quantitative high-throughput single-molecule RNA measurements for mammalian cells over a 2 x 2 mm2 region within an imaging depth of ~5 µm in less than 10 min and immunofluorescence imaging at a volumetric imaging rate of \u3e 30 Hz with significantly reduced light exposure. With implementation of an adaptive element, our microscope provides an extra degree of freedom in correcting aberrations induced by specimens and optical components, showing its capability of imaging thick specimens with high-fidelity of preserving volumetric information with fast imaging speed. We also demonstrate multimodal imaging that can be switched from 2.5DM to a 3D single-molecule localization imaging platform by encoding the depth information of each emitter into the shape of point spread function, which enables us to obtain a resolution of \u3c 50 nm. Our microscope offers multi-functional capability from fast volumetric high-throughput imaging, multi-color imaging to super-resolution imaging

    RNA AS A UNIQUE POLYMER TO BUILD CONTROLLABLE NANOSTRUCTURES FOR NANOMEDICINE AND NANOTECHNOLOGY

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    RNA nanotechnology is an emerging field that involves the design, construction and functionalization of nanostructures composed mainly of RNA for applications in biomedical and material sciences. RNA is a unique polymer with structural simplicity like DNA and functional diversity like proteins. A variety of RNA nanostructures have been reported with different geometrical structures and functionalities. This dissertation describes the design and construction of novel two-dimensional and three-dimensional self-assembled RNA nanostructures with applications in therapeutics delivery, cancer targeting and immunomodulation. Firstly, by using the ultra-stable pRNA three-way junction motif with controllable angles and arm lengths, tetrahedral architectures composed purely of RNA were successfully assembled via one-pot bottom-up assembly with high efficiency and thermal stability. By introducing arm sizes of 22 bp and 55 bp, two RNA tetrahedrons with similar global contour structure but with different sizes of 8 nm and 17 nm were successfully assembled. The RNA tetrahedrons were also highly amenable to functionalization. Fluorogenic RNA aptamers, ribozyme, siRNA, and protein-binding RNA aptamers were integrated into the tetrahedrons by simply fusing the respective sequences with the tetrahedral core modules. Secondly, I reported the design and construction of molecularly defined RNA cages with cube and dodecahedron shapes based on the stable pRNA 3WJ. The RNA cages can be easily self-assembled by single-step annealing. The RNA cages were further characterized by gel electrophoresis, cryo-electron microscopy and atomic force microscopy, confirming the spontaneous formation of the RNA cages. I also demonstrated that the constructed RNA cages could be used to deliver model drugs such as immunomodulatory CpG DNA into cells and elicit enhanced immune responses. Thirdly, by using the modular multi-domain strategy, molecular defined RNA nanowires can be successfully self-assembled via a bottom-up approach. Only four different 44-nucleotide single-stranded RNAs were used to assemble the RNA nanowire. The reported RNA nanowire has the potential to be explored in the future as the carrier for drug delivery or matrix for tissue engineering. Fourthly, the construction of RNA polygons for delivering immunoactive CpG oligonucleotides will be presented. When CpG oligonucleotides were incorporated into the RNA polygons, their immunomodulation effect for cytokine TNF-α and IL-6 induction was greatly enhanced, while RNA polygon controls induced unnoticeable cytokine induction. Moreover, the RNA polygons were delivered to macrophages specifically and the degree of immunostimulation greatly depended on the size, shape, and the number of payload per RNA polygon. Collectively, these findings demonstrated RNA nanotechnology can produce controllable nanostructures with different functionalities and result in potential applications in nanomedicine and nanotechnology

    Multiple layer image analysis for video microscopy

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    Motion analysis is a fundamental problem that serves as the basis for many other image analysis tasks, such as structure estimation and object segmentation. Many motion analysis techniques assume that objects are opaque and non-reflective, asserting that a single pixel is an observation of a single scene object. This assumption breaks down when observing semitransparent objects--a single pixel is an observation of the object and whatever lies behind it. This dissertation is concerned with methods for analyzing multiple layer motion in microscopy, a domain where most objects are semitransparent. I present a novel approach to estimating the transmission of light through stationary, semitransparent objects by estimating the gradient of the constant transmission observed over all frames in a video. This enables removing the non-moving elements from the video, providing an enhanced view of the moving elements. I present a novel structured illumination technique that introduces a semitransparent pattern layer to microscopy, enabling microscope stage tracking even in the presence of stationary, sparse, or moving specimens. Magnitude comparisons at the frequencies present in the pattern layer provide estimates of pattern orientation and focal depth. Two pattern tracking techniques are examined, one based on phase correlation at pattern frequencies, and one based on spatial correlation using a model of pattern layer appearance based on microscopy image formation. Finally, I present a method for designing optimal structured illumination patterns tuned for constraints imposed by specific microscopy experiments. This approach is based on analysis of the microscope's optical transfer function at different focal depths

    New methods for the study of Primary Ciliary Dyskinesia

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    Os cilios e flagelos são projeções celulares encontradas nas células eucariotas, são altamente conservados entre espécies e envolvidos na locomoção e movimentação de fluídos. A Discinésia Ciliar Primária (DCP) é uma doenca genética autossómica recessiva dos cílios móveis, que tem como consequência várias manifestações clínicas. Estima-se que a DCP afete ~1 em cada 10.000 pessoas, mas é mais prevalente em grupos com marcada consanguinidade. A DCP está associada até à data a mais de 40 genes causadores de doença. O diagnóstico da DCP envolve a combinação de vários testes, entre eles a microscopia electrónica (ME), teste determinante na classificação de anomalias ciliares. Neste trabalho foquei-me nos cílios móveis e em como se classificam as derivações à estrutura considerada normal. Este estudo levou ao desenvolvimento de feramentas e diretrizes que tornam o diagnóstico de DCP por EM mais estandardizado, informativo e fidedigno. A DCP necessita de ser modelada em organismos vertebrados como o ratinho, a rã e o peixe-zebra (PZ) para melhor conhecimento dos seus mecanismos moleculares. O PZ é um bom modelo de DCP porque apresenta diversos órgãos ciliados durante os estados larvares (cílios moveis e imoveis) e tem, até agora, homólogos de todos os genes causadores da doença humana. Desta forma a utilização de peixes mutantes tem sido um bom contributo para compreender esta doença humana. Neste trabalho investiguei por ME dois tipos de cílios móveis do PZ concluindo que estes apresentam semelhanças estruturais conservadas com os cílios móveis das vias aéreas do ser humano saudável e com DCP.Cilia and flagella are cellular protrusions found in eucaryotic cells, highly conserved between species and found in almost every cell type. Motile cilia are known for their motility properties and are involved in propelling and moving fluids. Primary ciliary dyskinesia (PCD) is an inherited autosomal-recessive disorder of motile cilia that results in several clinical manifestations. The estimated prevalence of PCD is ∼1 per 10,000 births, but it is more prevalent in populations where consanguinity is common, it is currently associated with mutations in more than 40 genes. To diagnose PCD it involves a combination of tests, in particular, electron microscopy (EM) that is essential for determining the type of ciliary ultrastructural defect. In this work I have focused on motile cilia ultrastructure and how the differences in cilia can be identified and classified, through the development of tools and guidelines to make the quantification and analysis of cilia more reliable and informative. The differential diagnosis of PCD is complex but crucial, and the development of new potential targeted treatments is essential. For better investigating the molecular mechanisms underlying PCD, it has been modelled in several organisms like mice, frogs and Zebrafish (ZF). ZF is a teleost vertebrate used in many areas of research, and a well-known animal model. ZF embryos develop quickly and allow unique advantages for research studies owing to their transparency during larval stages. ZF has many ciliated organs and presents primary cilia as well as motile cilia together with homologs for all the disease causing genes. The use of mutant zebrafish has been contributing to the better understanding of PCD molecular aetiology. Here, I investigated whether zebrafish cilia are ultrastructurally suitable for the study of PCD and concluded that the motile cilia of zebrafish resemble the cilia in the human airway in healthy conditions and in PCD

    Developing 3D novel edge detection and particle picking tools for electron tomography

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