subject:(Super resolution microscopy)

University of Cambridge

1. Kondo, Hanae. Spatio-temporal properties of membrane-localized actin nucleating complexes.

Degree: PhD, 2019, University of Cambridge

URL: https://www.repository.cam.ac.uk/handle/1810/289704

► The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at… (more)

▼ The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane.

Subjects/Keywords: actin; filopodia; super-resolution microscopy

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APA (6^th Edition):

Kondo, H. (2019). Spatio-temporal properties of membrane-localized actin nucleating complexes. (Doctoral Dissertation). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/289704

Chicago Manual of Style (16^th Edition):

Kondo, Hanae. “Spatio-temporal properties of membrane-localized actin nucleating complexes.” 2019. Doctoral Dissertation, University of Cambridge. Accessed January 20, 2021. https://www.repository.cam.ac.uk/handle/1810/289704.

MLA Handbook (7^th Edition):

Kondo, Hanae. “Spatio-temporal properties of membrane-localized actin nucleating complexes.” 2019. Web. 20 Jan 2021.

Vancouver:

Kondo H. Spatio-temporal properties of membrane-localized actin nucleating complexes. [Internet] [Doctoral dissertation]. University of Cambridge; 2019. [cited 2021 Jan 20]. Available from: https://www.repository.cam.ac.uk/handle/1810/289704.

Council of Science Editors:

Kondo H. Spatio-temporal properties of membrane-localized actin nucleating complexes. [Doctoral Dissertation]. University of Cambridge; 2019. Available from: https://www.repository.cam.ac.uk/handle/1810/289704

University of New South Wales

2. Baek, Jongho. Improvements in super-resolution fluorescence microscopy.

Degree: Medical Sciences, 2019, University of New South Wales

URL: http://handle.unsw.edu.au/1959.4/62228

► Resolution in fluorescence microscopy is one of the most critical parameters in cellular imaging. Super-resolution fluorescence microscopes have spatial resolutions below the diffraction limit (~250… (more)

▼ Resolution in fluorescence microscopy is one of the most critical parameters in cellular imaging. Super-resolution fluorescence microscopes have spatial resolutions below the diffraction limit (~250 nm) and are expected to revolutionise cell biology. Single molecule localization microscopy (SMLM) provides a molecular image of biological specimens while lattice light-sheet microscopy (LLSM) has the ability to provide 3D images and movies of live cells. The goal of this thesis is to explore how resolution can be improved via optical engineering solutions.First, an actively stabilized SMLM (termed Feedback SMLM) has developed that achieved localization precisions of 1-4 nm without any post-acquisition processing. The performance of the ‘Feedback SMLM’ was demonstrated with DNA origami structures and by imaging F-actin and signaling proteins in T cells. With this microscopy, it was possible to identify the width of 5-9 nm individual F-actin filaments and measure the separation distance of proteins below 20 nm. The Feedback SMLM instruments relies on non-fluorescent fiducial markers to correct the stage position during data acquisition. In Chapter 3, it was explored whether polystyrene beads could be replaced with photo-polymerized structures that are directly printed onto the glass coverslip. This has the advantage that the density and positioning of the fiducial markers can be controlled and the fiducial markers have a minimal impact on the biology under investigation. Here, we designed and printed different shapes of 3D fiducials via femtosecond laser photopolymerization, and demonstrated the potential of using these novel fiducials for stage stabilisation. Lattice light-sheet microscopy (LLSM) is a promising super-resolution technique that – unlike SMLM – works equally well in live and fixed samples. For Chapter 4, the installation and alignment of an LLSM instrument was performed, with which a resolution of 230 × 230 × 370 nm (xyz) was achieved. This LLSM setup was employed to obtain 3D images and movies of endocytosis, cancer spheroids and the structure of inflammasome.In conclusion, two different super-resolution microscopy techniques were explored. The localization precision of SMLM was improved by incorporating feedback loops into the set-up and the suitability of LLSM for dynamic imaging was tested on a range of applications. Advisors/Committee Members: Gaus, Katharina, Medical Sciences, Faculty of Medicine, UNSW.

Subjects/Keywords: Super-resolution fluorescence microscopy

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APA (6^th Edition):

Baek, J. (2019). Improvements in super-resolution fluorescence microscopy. (Doctoral Dissertation). University of New South Wales. Retrieved from http://handle.unsw.edu.au/1959.4/62228

Chicago Manual of Style (16^th Edition):

Baek, Jongho. “Improvements in super-resolution fluorescence microscopy.” 2019. Doctoral Dissertation, University of New South Wales. Accessed January 20, 2021. http://handle.unsw.edu.au/1959.4/62228.

MLA Handbook (7^th Edition):

Baek, Jongho. “Improvements in super-resolution fluorescence microscopy.” 2019. Web. 20 Jan 2021.

Vancouver:

Baek J. Improvements in super-resolution fluorescence microscopy. [Internet] [Doctoral dissertation]. University of New South Wales; 2019. [cited 2021 Jan 20]. Available from: http://handle.unsw.edu.au/1959.4/62228.

Council of Science Editors:

Baek J. Improvements in super-resolution fluorescence microscopy. [Doctoral Dissertation]. University of New South Wales; 2019. Available from: http://handle.unsw.edu.au/1959.4/62228

University of Texas – Austin

3. Weber, Maggie Leona. Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles.

Degree: PhD, Chemistry, 2015, University of Texas – Austin

URL: http://hdl.handle.net/2152/46540

► Surface-enhanced Raman scattering (SERS) from organic molecules is used to study how geometry affects plasmonic enhancement in noble metal nanoparticles using super-resolution analysis techniques coupled… (more)

▼ Surface-enhanced Raman scattering (SERS) from organic molecules is used to study how geometry affects plasmonic enhancement in noble metal nanoparticles using super-resolution analysis techniques coupled with scanning electron microscope (SEM) imaging. We demonstrate that super-resolution analysis can track emission from Raman-active molecules on nanoaggregate surfaces with resolution typically better than 5 nm, a significant improvement over the diffraction limit. This optical analysis technique is used to identify active junction regions, known as hot spots, in correlated SEM structural images of aggregated particles based on shape, size, and angular position of SERS emission. This correlated analysis is further extended to study the mechanism of silver nanoparticle luminescence and its positional relationship to SERS emission. These studies support the hypothesis that silver luminescence is enhanced via a different mechanism than SERS. Discrete dipole approximation calculations agree with experimental results, indicating that SERS emission is a product of local plasmonic enhancement in specific junction regions between nanoparticles whereas silver luminescence is dependent upon nanoaggregate geometry and the collective plasmon modes within a structure. These theoretical calculations of luminescence centroid location can assist with SERS-active junction region assignment in higher order aggregates (e.g. trimers, tetramers, etc.) Finally, correlated optical and structural studies are used to develop understanding of site-specific electrochemical potentials on silver and gold nanoparticle aggregates using a redox active Raman reporter molecule. We demonstrate that there is a weak correlation between emission centroid location and bulk sample potential. Silver colloid studies demonstrate variability in modulation behavior and are highly unpredictable, whereas gold colloids with the redox molecule covalently tethered to their surface via a gold-thiol bond show more reproducible modulation. Furthermore, these tethered studies demonstrate geometrical agreement between site-specific emission at negative potentials and junction regions in nanoaggregate SEM images indicating that plasmonic enhancement may affect local nanoparticle surface potentials. Advisors/Committee Members: Brodbelt, Jennifer S. (advisor), Willets, Katherine A. (advisor), Hoffman, David W (committee member), Korgel, Brian A (committee member), Roberts, Sean T (committee member), Anslyn, Aric V (committee member), Holcombe, James A (committee member).

Subjects/Keywords: Raman; Nanoparticles; Super-resolution; Microscopy

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Weber, M. L. (2015). Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles. (Doctoral Dissertation). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/46540

Chicago Manual of Style (16^th Edition):

Weber, Maggie Leona. “Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles.” 2015. Doctoral Dissertation, University of Texas – Austin. Accessed January 20, 2021. http://hdl.handle.net/2152/46540.

MLA Handbook (7^th Edition):

Weber, Maggie Leona. “Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles.” 2015. Web. 20 Jan 2021.

Vancouver:

Weber ML. Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles. [Internet] [Doctoral dissertation]. University of Texas – Austin; 2015. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2152/46540.

Council of Science Editors:

Weber ML. Using correlated super-resolution optical and structural studies to investigate surface-enhanced Raman scattering on silver & gold nanoparticles. [Doctoral Dissertation]. University of Texas – Austin; 2015. Available from: http://hdl.handle.net/2152/46540

University of Utah

4. Ebeling, Carl G. Increasing estimation precision in localization microscopy.

Degree: PhD, Physics & Astronomy, 2015, University of Utah

URL: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/3746/rec/1344

► This dissertation studies detection-based methods to increase the estimation precision of single point-source emitters in the field of localization microscopy. Localization microscopy is a novel… (more)

▼ This dissertation studies detection-based methods to increase the estimation precision of single point-source emitters in the field of localization microscopy. Localization microscopy is a novel method allowing for the localization of optical point-source emitters below the Abbe diffraction limit of optical microscopy. This is accomplished by optically controlling the active, or bright, state of individual molecules within a sample. The use of time-multiplexing of the active state allows for the temporal and spatial isolation of single point-source emitters. Isolating individual sources within a sample allows for statistical analysis on their emission point-spread function profile, and the spatial coordinates of the point-source may be discerned below the optical response of the microscope system. Localization microscopy enables the identification of individual point-source emitter locations approximately an order of magnitude below standard, diffraction-limited optical techniques. The precision of localization microscopy methods is limited by the statistical uncertainty in which the location of these sources may be estimated. By utilizing a detection- based interferometer, an interference pattern may be super-imposed over the emission signal. Theoretical analysis and Monte-Carlo simulations by means of Fisher information theory demonstrate that the incorporation of a modulation structure over the emission signal allow for a more precise estimation when compared to conventional localization methods for the same number of recorded photons. These theoretical calculation and simulations are demonstrated through the use of two proof-of-concept experiments utilizing a modified Mach-Zehnder interferometer. The first methodology improves the localization precision of a single nanoparticle over the theoretical limit for an Airy-disk point-spread function by using self-interference to spatially modulate the recorded point-spread function. Experimental analysis demonstrates an improvement factor of ~3 to 5 over conventional localization methods. A related method employs the phase induced onto the Fourier domain signal due to path length differences in the Mach-Zehnder interferometer to improve localization precision. The localization capability of a modified Fourier domain signal generated by self-interference is utilized to yield a two-fold improvement in the localization precision for a given number of photons compared to a standard Gaussian intensity distribution of the corresponding point-spread function.

Subjects/Keywords: Estimation precision; Interference; Localization microscopy; Super resolution

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APA (6^th Edition):

Ebeling, C. G. (2015). Increasing estimation precision in localization microscopy. (Doctoral Dissertation). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/3746/rec/1344

Chicago Manual of Style (16^th Edition):

Ebeling, Carl G. “Increasing estimation precision in localization microscopy.” 2015. Doctoral Dissertation, University of Utah. Accessed January 20, 2021. http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/3746/rec/1344.

MLA Handbook (7^th Edition):

Ebeling, Carl G. “Increasing estimation precision in localization microscopy.” 2015. Web. 20 Jan 2021.

Vancouver:

Ebeling CG. Increasing estimation precision in localization microscopy. [Internet] [Doctoral dissertation]. University of Utah; 2015. [cited 2021 Jan 20]. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/3746/rec/1344.

Council of Science Editors:

Ebeling CG. Increasing estimation precision in localization microscopy. [Doctoral Dissertation]. University of Utah; 2015. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/3746/rec/1344

University of Manchester

5. Oszmiana, Anna. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.

Degree: 2016, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300569

► Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed… (more)

▼ Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed or up-regulated on cancerous or infected cells, while inhibitory receptors bind self-molecules to induce tolerance against healthy cells. Highly homologous pairs of activating and inhibitory receptors are also expressed on NK cells, including Killer Ig-like Receptors KIR2DL1 and KIR2DS1, which bind the same ligands, class I MHC proteins from the C2 group. Here, two super-resolution microscopy techniques, stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecule return (GSDIM) were used to examine the nanometre-scale organization of KIR2DL1 and KIR2DS1, as well as molecules engaged in their signalling.Both receptors were observed to constitutively assemble in nanometre-scale clusters at the surface of NK cells but displayed differential patterns of clustering - the activating receptor KIR2DS1 formed nanoclusters 2.3-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters was controlled by transmembrane amino-acid 233, a lysine in KIR2DS1. Mutated variant of KIR2DS1 in which lysine 233 was substituted with alanine formed significantly smaller clusters than the wild-type KIR2DS1. Reciprocally, substitution of isoleucine found at position 233 in KIR2DL1 sequence with lysine resulted in the receptor assembling into larger clusters.Super-resolution microscopy also revealed two ways in which KIR nanoclusters impact signalling. First, KIR2DS1 and DAP12 nanoclusters were juxtaposed in the resting-cell state but coalesced upon receptor ligation. Second, quantitative super-resolution microscopy revealed that membrane-proximal clusters of the kinase ZAP-70 or phosphatase SHP-1, as well as their phosphorylated active forms, were more often found in contact with larger KIR nanoclusters.Together, this work has established that size of KIR nanoclusters depends on the transmembrane sequence and impacts downstream signalling. Advisors/Committee Members: HUSSELL, TRACY T, Davis, Daniel, Hussell, Tracy.

Subjects/Keywords: Natural Killer cells; Super-resolution microscopy

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Oszmiana, A. (2016). Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300569

Chicago Manual of Style (16^th Edition):

Oszmiana, Anna. “Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300569.

MLA Handbook (7^th Edition):

Oszmiana, Anna. “Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.” 2016. Web. 20 Jan 2021.

Vancouver:

Oszmiana A. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 20]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300569.

Council of Science Editors:

Oszmiana A. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. [Doctoral Dissertation]. University of Manchester; 2016. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300569

University of Manchester

6. Oszmiana, Anna. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.

Degree: PhD, 2016, University of Manchester

URL: https://www.research.manchester.ac.uk/portal/en/theses/nanometrescale-organization-of-the-natural-killer-cell-receptors-kir2dl1-and-kir2ds1-and-its-implications-for-signalling(218abc3c-4408-49b8-aa69-f68d1832e726).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740284

► Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed… (more)

▼ Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed or up-regulated on cancerous or infected cells, while inhibitory receptors bind self-molecules to induce tolerance against healthy cells. Highly homologous pairs of activating and inhibitory receptors are also expressed on NK cells, including Killer Ig-like Receptors KIR2DL1 and KIR2DS1, which bind the same ligands, class I MHC proteins from the C2 group. Here, two super-resolution microscopy techniques, stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecule return (GSDIM) were used to examine the nanometre-scale organization of KIR2DL1 and KIR2DS1, as well as molecules engaged in their signalling. Both receptors were observed to constitutively assemble in nanometre-scale clusters at the surface of NK cells but displayed differential patterns of clustering - the activating receptor KIR2DS1 formed nanoclusters 2.3-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters was controlled by transmembrane amino-acid 233, a lysine in KIR2DS1. Mutated variant of KIR2DS1 in which lysine 233 was substituted with alanine formed significantly smaller clusters than the wild-type KIR2DS1. Reciprocally, substitution of isoleucine found at position 233 in KIR2DL1 sequence with lysine resulted in the receptor assembling into larger clusters. Super-resolution microscopy also revealed two ways in which KIR nanoclusters impact signalling. First, KIR2DS1 and DAP12 nanoclusters were juxtaposed in the resting-cell state but coalesced upon receptor ligation. Second, quantitative super-resolution microscopy revealed that membrane-proximal clusters of the kinase ZAP-70 or phosphatase SHP-1, as well as their phosphorylated active forms, were more often found in contact with larger KIR nanoclusters. Together, this work has established that size of KIR nanoclusters depends on the transmembrane sequence and impacts downstream signalling.

Subjects/Keywords: 616.07; Natural Killer cells; Super-resolution microscopy

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Oszmiana, A. (2016). Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/nanometrescale-organization-of-the-natural-killer-cell-receptors-kir2dl1-and-kir2ds1-and-its-implications-for-signalling(218abc3c-4408-49b8-aa69-f68d1832e726).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740284

Chicago Manual of Style (16^th Edition):

Oszmiana, Anna. “Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021. https://www.research.manchester.ac.uk/portal/en/theses/nanometrescale-organization-of-the-natural-killer-cell-receptors-kir2dl1-and-kir2ds1-and-its-implications-for-signalling(218abc3c-4408-49b8-aa69-f68d1832e726).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740284.

MLA Handbook (7^th Edition):

Oszmiana, Anna. “Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling.” 2016. Web. 20 Jan 2021.

Vancouver:

Oszmiana A. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 20]. Available from: https://www.research.manchester.ac.uk/portal/en/theses/nanometrescale-organization-of-the-natural-killer-cell-receptors-kir2dl1-and-kir2ds1-and-its-implications-for-signalling(218abc3c-4408-49b8-aa69-f68d1832e726).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740284.

Council of Science Editors:

Oszmiana A. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling. [Doctoral Dissertation]. University of Manchester; 2016. Available from: https://www.research.manchester.ac.uk/portal/en/theses/nanometrescale-organization-of-the-natural-killer-cell-receptors-kir2dl1-and-kir2ds1-and-its-implications-for-signalling(218abc3c-4408-49b8-aa69-f68d1832e726).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740284

University of Manchester

7. Sanders, James Henry. Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures.

Degree: PhD, 2015, University of Manchester

URL: https://www.research.manchester.ac.uk/portal/en/theses/direct-stochastic-optical-reconstruction-microscopy-dstorm-imaging-of-cellular-structures(915e2c88-c81a-4b24-ac53-6ab7ffcbf4d8).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647388

► The diffraction limit restricts conventional light microscopes to approximately 250 nm laterally and 500 nm axially, these limits being first proposed by Abbe in 1873.… (more)

▼ The diffraction limit restricts conventional light microscopes to approximately 250 nm laterally and 500 nm axially, these limits being first proposed by Abbe in 1873. Despite this, optical microscopes have found many applications in biological research and single cells that are 10 - 100 um in size. Furthermore by coupling the non-invasive nature of a light microscope with highly sensitive fluorescent probes, fluorescence microscopy has also become a standard imaging technique. Recent advances in fluorescence microscopy now provide a number of methods to circumvent the Abbe diffraction limit, with many techniques becoming prevalent over the last 10 years including direct Stochastic Optical Reconstruction Microscopy (dSTORM). A dSTORM system has been constructed and calibrated using a commercially available inverted florescence microscope and total internal reflection florescence (TIRF) imaging. dSTORM relies on the ability to switch sparse subsets of fluorophores and temporally separate them. Provided the spatial separation is sufficient between any member of a subset, the average error with which the emission can be localized is much less than size of the emission profile itself. The underlying mechanism for this switching is detailed based on the principle of photoinduced electron transfer (PET). The switching characteristics of the common florescent dye Alexa Fluor 568 are investigated and shown to be controlled by a number of factors including the excitation intensity and concentration of the primary thiol cysteamine beta-MEA. A number of parameters are defined, including the dye switching rate, for a given set of physical parameters. U2OS cells are labelled for the microtubule protein Tubulin using immunofluorescent labelling strategies. A direct comparison is made between diffraction limited TIRF images and dSTORM reconstructed images, with an average width for microtubules determined to (58.2 ± 8.1) nm. Further measurements are made by labelling the Rab5 effector Early Endosome Antigen 1 (EEA1). From this the aspect ratio for early endosomes is determined to be 1.68 ± 0.7 with an average radius of (45.8 ± 18.8) nm. The point spatial distribution of EEA1 is investigated by using the linearised form of Ripley's K-function H(r) and the null hypothesis of complete spatial randomness tested. EEA1 is shown to cluster at radius of 58.7 nm on individual endosomes, thought to be due to the well defined binding domains present on early endosomes for EEA1. Further evidence suggests that clustering is also exhibited at another maximum of approximately 500 nm when looking at an ensemble of EEA1 and early endosomes.

Subjects/Keywords: 621.36; Super-resolution; dSTORM; fluorescence microscopy

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APA (6^th Edition):

Sanders, J. H. (2015). Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/direct-stochastic-optical-reconstruction-microscopy-dstorm-imaging-of-cellular-structures(915e2c88-c81a-4b24-ac53-6ab7ffcbf4d8).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647388

Chicago Manual of Style (16^th Edition):

Sanders, James Henry. “Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures.” 2015. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021. https://www.research.manchester.ac.uk/portal/en/theses/direct-stochastic-optical-reconstruction-microscopy-dstorm-imaging-of-cellular-structures(915e2c88-c81a-4b24-ac53-6ab7ffcbf4d8).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647388.

MLA Handbook (7^th Edition):

Sanders, James Henry. “Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures.” 2015. Web. 20 Jan 2021.

Vancouver:

Sanders JH. Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures. [Internet] [Doctoral dissertation]. University of Manchester; 2015. [cited 2021 Jan 20]. Available from: https://www.research.manchester.ac.uk/portal/en/theses/direct-stochastic-optical-reconstruction-microscopy-dstorm-imaging-of-cellular-structures(915e2c88-c81a-4b24-ac53-6ab7ffcbf4d8).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647388.

Council of Science Editors:

Sanders JH. Direct stochastic optical reconstruction microscopy (dSTORM) imaging of cellular structures. [Doctoral Dissertation]. University of Manchester; 2015. Available from: https://www.research.manchester.ac.uk/portal/en/theses/direct-stochastic-optical-reconstruction-microscopy-dstorm-imaging-of-cellular-structures(915e2c88-c81a-4b24-ac53-6ab7ffcbf4d8).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647388

University of Texas – Austin

8. -9009-1481. Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy.

Degree: PhD, Biomedical Engineering, 2020, University of Texas – Austin

URL: http://dx.doi.org/10.26153/tsw/8974

► Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in… (more)

▼ Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging. Advisors/Committee Members: Dunn, Andrew Kenneth, 1970- (advisor), Harris, Kristen (committee member), Milner, Thomas (committee member), Yeh, Hsin-Chih (Tim) (committee member).

Subjects/Keywords: Multiphoton microscopy; Ultrafast lasers; Super-resolution optics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

-9009-1481. (2020). Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy. (Doctoral Dissertation). University of Texas – Austin. Retrieved from http://dx.doi.org/10.26153/tsw/8974

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

Chicago Manual of Style (16^th Edition):

-9009-1481. “Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy.” 2020. Doctoral Dissertation, University of Texas – Austin. Accessed January 20, 2021. http://dx.doi.org/10.26153/tsw/8974.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

MLA Handbook (7^th Edition):

-9009-1481. “Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy.” 2020. Web. 20 Jan 2021.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

Vancouver:

-9009-1481. Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy. [Internet] [Doctoral dissertation]. University of Texas – Austin; 2020. [cited 2021 Jan 20]. Available from: http://dx.doi.org/10.26153/tsw/8974.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

Council of Science Editors:

-9009-1481. Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy. [Doctoral Dissertation]. University of Texas – Austin; 2020. Available from: http://dx.doi.org/10.26153/tsw/8974

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

9. Wang, Ruixing. STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique.

Degree: Docteur es, Microbiologie, 2018, Aix Marseille Université

URL: http://www.theses.fr/2018AIXM0163

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Les techniques de super-résolution offrent un nouvel aperçu de la description de l'organisation moléculaire dynamique de la membrane plasmique. Parmi ces techniques, la microscopie par… (more)

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Les techniques de super-résolution offrent un nouvel aperçu de la description de l'organisation moléculaire dynamique de la membrane plasmique. Parmi ces techniques, la microscopie par déplétion d'émission stimulée (stimulated emission depletion, STED) dépasse la limite de diffraction optique et atteint une résolution de quelques dizaines de nanomètres. Il est une technique polyvalente qui peut être combinée avec d'autres techniques telles que la spectroscopie par corrélation de fluorescence (fluorescence correlation spectroscopy, FCS), fournissant des résolutions spatiales et temporelles élevées pour explorer les processus dynamiques qui se produisent dans les cellules vivantes. Ce projet de doctorat vise à mettre en œuvre un microscope STED, puis à combiner ce module STED avec la technique FCS pour les applications biologiques. Des études théoriques du STED et de la technique combinant STED et FCS ont permis dans les aspects spatio-temporels. Une solution analytique pour la fonction d'autocorrélation FCS a été dérivée dans l'état de déplétion STED incomplet. et un nouveau modèle d'ajustement FCS a été proposé. La méthode de variation du volume d’observation FCS (spot variation FCS, svFCS) a démontré sa capacité à identifier la présence de nanodomaines limitant la diffusion latérale des molécules dans la membrane plasmique. L’approche STED-FCS permet d’étendre l’application de la svFCS à l'échelle nanométrique afin d’évaluer la persistance plus ou moins importante de tels nanodomaines. Dans ce contexte, des simulations préliminaires de Monte Carlo ont été réalisées figurant des molécules diffusant en présence d'auto-assemblage/désassemblage dynamique des nanodomaines.

Super-resolution techniques offer new insight into the description of the dynamic molecular organization at the plasma membrane. Among these techniques, the stimulated emission depletion (STED) microscopy breaks the optical diffraction limit and reaches the resolution of tens of nanometer. It is a versatile setup that can be combined with other techniques such as fluorescence correlation spectroscopy (FCS), providing both high spatial and temporal resolutions to explore dynamic processes occurring in live cells. This PhD project aims at implementing a STED microscope, and then at combining this STED module with FCS technique for biological applications. Detailed theoretical studies on STED and the combined STED-FCS technique in spatio-temporal aspects were performed. An analytical solution for FCS autocorrelation function was derived in the condition of incomplete STED depletion and a new FCS fitting model was proposed to overcome this problem. The spot variation FCS (svFCS) method has demonstrated its capability to identify the presence of nanodomains constraining the lateral diffusion of molecules at the plasma membrane. The STED-FCS can extend the svFCS approach to the nanoscale evaluating the long-lasting existence of such nanodomains. Within this frame, preliminary Monte Carlo simulations were conducted mimicking molecules diffusing in the…

Advisors/Committee Members: Marguet, Didier (thesis director), Rigneault, Hervé (thesis director).

Subjects/Keywords: Sted; Fcs; Super-Résolution; Microscopie à fluorescence; Sted; Fcs; Super-Resolution; Fluorescence microscopy; 579

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Wang, R. (2018). STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique. (Doctoral Dissertation). Aix Marseille Université. Retrieved from http://www.theses.fr/2018AIXM0163

Chicago Manual of Style (16^th Edition):

Wang, Ruixing. “STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique.” 2018. Doctoral Dissertation, Aix Marseille Université. Accessed January 20, 2021. http://www.theses.fr/2018AIXM0163.

MLA Handbook (7^th Edition):

Wang, Ruixing. “STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique.” 2018. Web. 20 Jan 2021.

Vancouver:

Wang R. STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique. [Internet] [Doctoral dissertation]. Aix Marseille Université 2018. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2018AIXM0163.

Council of Science Editors:

Wang R. STED-fluorescence correlation spectroscopy for dynamic observations in cell biology : from theoretical to practical approaches : STED-spectroscopie de corrélation de fluorescence pour des observations dynamiques en biologie cellulaire : de l'approche théorique à l'approche pratique. [Doctoral Dissertation]. Aix Marseille Université 2018. Available from: http://www.theses.fr/2018AIXM0163

10. Yang, Bin. New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution.

Degree: Docteur es, Lasers, matière et nanosciences, 2015, Bordeaux

URL: http://www.theses.fr/2015BORD0075

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La première méthode vise à améliorer la vitesse d’imagerie de la microscopie super-résolue àtempérature ambiante pour des applications biologiques. En tant qu’une technique de scan,… (more)

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La première méthode vise à améliorer la vitesse d’imagerie de la microscopie super-résolue àtempérature ambiante pour des applications biologiques. En tant qu’une technique de scan, lamicroscopie STED a besoins d’être parallélisé pour faire de l’imagerie rapide en champ large. Nousavons obtenu une parallélisation massive de la microscopie STED en utilisant les réseaux d’optiqueavec une excitation en en champ large et une caméra rapide pour détection. Les images super-résoluesd’un champ de 3 μm par 3 μm sont acquises en scannant une maille élémentaire du réseau optique, quipeut être aussi petite que 290 nm * 290 nm. La microscopie Lattice-STED est démontrée avec unerésolution allant jusqu'à 70 nm à une cadence de 12,5 images par seconde.La deuxième méthode étend la microscopie super-résolue à la température de l’hélium liquide pourdes applications aux technologies quantiques. Des résolutions optiques à l'échelle nanométrique desémetteurs quantique est une étape cruciale vers le contrôle des états délocalisés formés par lesinteractions fortes et cohérentes entre des émetteurs. Dans ce contexte, nous avons développé unetechnique de microscopie à des températures cryogéniques, dénommée la microscopie Essat. Cettetechnique est basée sur la saturation optique de l'état excité des molécules fluorescentes uniques parl’excitation d’un faisceau en forme d’anneau. Une résolution moins de 10 nm est obtenue avec debasses intensités d'excitation, plus de millions de fois plus faibles que celles utilisées dans lamicroscopie STED à la température ambiante. Par rapport aux approches basées sur la superlocalisation,notre technique offre une occasion unique de résoudre sous la limite de diffraction lesmolécules uniques ayant des fréquences de résonance optiques qui se chevauchent. Ceci ouvre la voieà l'étude des interactions cohérentes entre émetteurs uniques et à la manipulation de leur degréd'intrication.

The first technique aims at improving the imaging speed of super-resolution microscopy at roomtemperature for biological applications. As a scanning technique, STED (Stimulated EmissionDepletion) microscopy needs parallelization for fast wide-field imaging. Using well-designed opticallattices for depletion together with wide-field excitation and a fast camera for detection, we achievelarge parallelization of STED microscopy. Wide field of view super-resolved images are acquired byscanning over a single unit cell of the optical lattice, which can be as small as 290 nm * 290 nm.Lattice-STED imaging is demonstrated with a resolution down to 70 nm at 12.5 frames per second.The second one extends super-resolution microscopy to liquid helium temperature for applications inquantum technologies. Optical resolution of solid-state single quantum emitters at the nanometer scaleis a challenging step towards the control of delocalized states formed by strongly and coherentlyinteracting emitters. ESSat (Excited State Saturation) microscopy operating at cryogenic temperaturesis based on optical saturation of the excited state of single…

Advisors/Committee Members: Lounis, Brahim (thesis director).

Subjects/Keywords: Super-résolution; Microscopie; STED; Molécule unique; Super-resolution; Microscopy; STED; Single molecule

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Yang, B. (2015). New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution. (Doctoral Dissertation). Bordeaux. Retrieved from http://www.theses.fr/2015BORD0075

Chicago Manual of Style (16^th Edition):

Yang, Bin. “New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution.” 2015. Doctoral Dissertation, Bordeaux. Accessed January 20, 2021. http://www.theses.fr/2015BORD0075.

MLA Handbook (7^th Edition):

Yang, Bin. “New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution.” 2015. Web. 20 Jan 2021.

Vancouver:

Yang B. New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution. [Internet] [Doctoral dissertation]. Bordeaux; 2015. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2015BORD0075.

Council of Science Editors:

Yang B. New approaches in super-resolution microscopy : Nouvelles approches microscope de super-résolution. [Doctoral Dissertation]. Bordeaux; 2015. Available from: http://www.theses.fr/2015BORD0075

11. Berardozzi, Romain. Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy.

Degree: Docteur es, Biologie structurale et nanobiologie, 2016, Université Grenoble Alpes (ComUE)

URL: http://www.theses.fr/2016GREAV015

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La microscopie de super-résolution PALM (microscopie de localisation après photo-activation) est un outil performant pour l'étude des cellules à l'échelle nanométrique. Dans ses applications avancées,… (more)

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La microscopie de super-résolution PALM (microscopie de localisation après photo-activation) est un outil performant pour l'étude des cellules à l'échelle nanométrique. Dans ses applications avancées, la microscopie PALM permet l'étude quantitative et dynamique des objets et événements biologiques. Ces applications sont cependant limitées par le comportement photophysique complexe des protéines fluorescentes photoconvertibles vert à rouge (PCFPs) utilisées comme marqueurs. En particulier, les transitions répétées et stochastiques des PCFPs entre un état sombre et un état fluorescent (scintillement) ainsi que l'incomplétude de photoconversion compliquent l'extraction d'informations quantitatives.Nos travaux combinant cristallographie aux rayons X des protéines et microscopie de localisation ont permis de mettre en évidence le rôle central d'un acide aminé conservé au sein des PCFPs, l'arginine 66, dans le contrôle du scintillement et du photoblanchiment de la forme rouge de deux PCFPs populaires: mEos2 et Dendra2.D'autre part, des résultats préliminaires suggèrent que dans leur formes vertes et dans les conditions d'illumination classiques PALM, les PCFPs entrent dans un état sombre de long temps de vie ce qui ralentit la photoconversion.Nos résultats ouvrent la porte à la conception raisonnée de nouvelles PCFPs optimisées pour les applications quantitatives et dynamiques du PALM.

Super-resolution PALM microscopy (photoactivated localization microscopy) is a powerful tool to investigate the cells with nanoscopic accuracy. Advanced PALM microscopy allows to quantitatively and dynamically study biological objects and events. These applications are nevertheless limited by the complex photophysical behavior of the green-to-red photoconvertible fluorescent proteins (PCFPs) used as markers. In particular, PCFPs red forms repeated and stochastic transitions between a fluorescent and a dark state (blinking) as well as photoconversion uncompleteness complicate the extraction of quantitative information.Our study, by combining X-ray crystallography and localization microscopy, evidences that a single aminoacid well conserved among PCFPs, the arginine 66, controls the blinking and photobleaching behavior of two popular PCFPs: mEos2 and Dendra2.Preliminary results suggest that in their green forms and under PALM classical illumination conditions, PCFPs switch to a long-lived dark state resulting in a photoconversion slowing down.Our results open the door to future rational engineering of enhanced PCFPs for quantitative and dynamic PALM.

Advisors/Committee Members: Bourgeois, Dominique (thesis director).

Subjects/Keywords: Phototransformation; Microscopie super-Résolution; Protéines fluorescentes; Phototransformation; Super-Resolution microscopy; Fluorescent proteins; 570

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Berardozzi, R. (2016). Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy. (Doctoral Dissertation). Université Grenoble Alpes (ComUE). Retrieved from http://www.theses.fr/2016GREAV015

Chicago Manual of Style (16^th Edition):

Berardozzi, Romain. “Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy.” 2016. Doctoral Dissertation, Université Grenoble Alpes (ComUE). Accessed January 20, 2021. http://www.theses.fr/2016GREAV015.

MLA Handbook (7^th Edition):

Berardozzi, Romain. “Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy.” 2016. Web. 20 Jan 2021.

Vancouver:

Berardozzi R. Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy. [Internet] [Doctoral dissertation]. Université Grenoble Alpes (ComUE); 2016. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2016GREAV015.

Council of Science Editors:

Berardozzi R. Etude photophysique des protéines fluorescentes photoconvertibles utilisées en microscopie de super-résolution : Photophysical study of photoconvertible fluorescent proteins used as markers in super-resolution microscopy. [Doctoral Dissertation]. Université Grenoble Alpes (ComUE); 2016. Available from: http://www.theses.fr/2016GREAV015

12. Baptiste, Amouroux. Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy.

Degree: Docteur es, Chimie macromoléculaire et supramoléculaire, 2019, Université Toulouse III – Paul Sabatier

URL: http://www.theses.fr/2019TOU30106

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Les nanoparticules Upconverting (UCNP) à base de lanthanides possèdent la propriété fascinante d’être capables de convertir des photons infrarouges en photons de plus haute énergie… (more)

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Les nanoparticules Upconverting (UCNP) à base de lanthanides possèdent la propriété fascinante d’être capables de convertir des photons infrarouges en photons de plus haute énergie sans recourir à des fluences de laser élevées. Ce décalage Anti-Stokes conduit à rapport signal sur bruit meilleur que pour la luminescence classique. Associé à leur photostabilité (non clignotantes, non photolysables), un spectre d’émission indépendant de leur taille, une faible toxicité, ces matériaux inorganiques sont devenus un outil de choix en biologie, en particulier en imagerie biologique, à côté des Quantum Dots. Cependant, l’émission globale s’effondre rapidement quand la taille des UCNP est réduite. En conséquence la réalisation de particules ultra petites et efficaces reste un défi. Le présent mémoire s’intéresse au design de structures nanohybrides fondées sur des particules ultra petites de NaREF4, avec pour objectif la microscopie super-résolue. Le travail s’est organisé en trois phases. Tout d’abord nous avons étudié la réduction en taille des UCNP de 10-20 nm à moins de 5 nm, en se focalisant sur leur composition et l’amélioration du procédé de synthèse. En particulier nous avons montré l’importance de la conjonction du processus de mélange avec la conduite de l’étape à haute température. Pour cette dernière l’emploi du chauffage micro-onde, avec un cyclage en température original a permis de contrôler efficacement le mûrissement d’Ostwald. La librairie de particules ainsi construite a permis d’étudier la photophysique des processus de redistribution de l’énergie au sein des particules sur des montages « maison » ou des équipements dédiés grâce à des collaborations avec des équipes de Berlin ou Lille. Il en ressort que les quantificateurs usuels de luminescence sont inadéquats pour décrire le phénomène d’upconversion. Aussi avons-nous débuté l’élaboration d’un modèle cinétique approprié. Enfin, la construction de nanohybrides impliquant un polymère photochrome hydrophile a été explorée. La sélection du colorant approprié et les premières études photocinétiques ont été menées. Cette approche couplant nos « nanolampes » avec un « volet photochrome » a pour but de proposer une alternative innovante au développement de la super-résolution par STED.

Lanthanide-based Upconverting nanoparticles (UCNPs) show the fascinating property of converting low energy NIR photons into higher energy ones without requiring high laser fluences. This unique large anti-Stokes shift affords a higher signal-to-noise ratio than standard luminescent compounds. Associated to their photostability (non-blinking, non-bleaching), their size-independent emission spectrum and a limited toxicity, these inorganic materials have become an interesting tool in Biology besides Quantum Dots, especially for Bioimaging. However, the overall emission plummets sharply when the size is reduced. Therefore, efficient ultrasmall UCNPs are still challenging to obtain. The present work is dedicated to the design of innovative nanohybrid structures based on NaREF4 with…

Advisors/Committee Members: Coudret, Christophe (thesis director), Gauffre, Fabienne (thesis director).

Subjects/Keywords: Upconversion; Photochimie; Polymère; Nanohybride; Microscopie super-résolution; Upconversion; Photochemistry; Polymer; Nanohybrid; Super-resolution microscopy

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Baptiste, A. (2019). Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy. (Doctoral Dissertation). Université Toulouse III – Paul Sabatier. Retrieved from http://www.theses.fr/2019TOU30106

Chicago Manual of Style (16^th Edition):

Baptiste, Amouroux. “Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy.” 2019. Doctoral Dissertation, Université Toulouse III – Paul Sabatier. Accessed January 20, 2021. http://www.theses.fr/2019TOU30106.

MLA Handbook (7^th Edition):

Baptiste, Amouroux. “Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy.” 2019. Web. 20 Jan 2021.

Vancouver:

Baptiste A. Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy. [Internet] [Doctoral dissertation]. Université Toulouse III – Paul Sabatier; 2019. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2019TOU30106.

Council of Science Editors:

Baptiste A. Nanoparticules upconverting : vers la microscopie en super-résolution : Upconversion nanoparticles : towards super-resolution microscopy. [Doctoral Dissertation]. Université Toulouse III – Paul Sabatier; 2019. Available from: http://www.theses.fr/2019TOU30106

13. Cabriel, Clément. Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie.

Degree: Docteur es, Optique et photonique, 2019, Université Paris-Saclay (ComUE)

URL: http://www.theses.fr/2019SACLS220

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Pour analyser la structure et la dynamique des échantillons, la biologie cellulaire repose sur l'utilisation d'outils d'imagerie. En particulier, la microscopie de fluorescence offre une… (more)

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Pour analyser la structure et la dynamique des échantillons, la biologie cellulaire repose sur l'utilisation d'outils d'imagerie. En particulier, la microscopie de fluorescence offre une grande spécificité et une toxicité réduite. L'émergence récente des méthodes de super-résolution a permis d'outrepasser la limite de diffraction et ouvert de nouvelles perspectives d'études. Les stratégies de molécule unique sont particulièrement adaptées à l'imagerie nanométrique tridimensionnelle, et permettent de nombreux couplages avec des modalités complémentaires ; toutefois, leur manque de reproductibilité entrave leur généralisation.Nous proposons ici de nouvelles méthodes dans le but de remédier à ces problèmes en facilitant leur application en biologie cellulaire, en chimie et en science des matériaux. Tout d'abord, nous présentons des protocoles et échantillons dédiés aux acquisitions de calibration et de mesure de performances. Nous décrivons également plusieurs exemples d'utilisation de super-localisation tridimensionnelle dans le cadre d'études d'adhésion cellulaire et de résistance bactérienne.Ensuite, nous nous concentrons au développement d'une nouvelle méthode de microscopie de localisation de molécules uniques tri-dimensionnelle permettant l'élimination de biais de détection. Ceci est permis par le couplage entre deux stratégies complémentaires: la mise en forme de fonction d'étalement de point, et la détection de la fluorescence d'angle super-critique. L'intercorrélation et la recombinaison des informations latérales et axiales permet l'obtention d'une résolution quasi-isotrope, avec des précisions jusqu'à 15 nanomètres sur une plage de capture d'un micron. Nous mettons en évidence l'insensibilité de la méthode aux biais d'imagerie comme la dérive axiale, l'aberration chromatique et l'inclinaison de l'échantillon, et nous l'illustrons à travers des applications à la neurobiologie et au marquage de bactéries.Pour finir, nous présentons deux nouvelles approches pour le découplage d'acquisitions multi-espèces simultanées. Toutes deux basées entièrement sur le post-traitement des données acquises, elles exploitent respectivement la mesure des tailles des taches et le comportement dynamique du clignotement. Après une preuve de principe, nous évaluons l'impact des différents paramètres susceptibles d'influencer les résultats. Nous concluons en proposant des pistes d'amélioration des performances de découplage, et en suggérant de possibles couplages avec des méthodes complémentaires en imagerie de molécules uniques.

Cell biology relies on imaging tools to provide structural and dynamic information about samples. Among them, fluorescence microscopy offers a compromise between high specificity and low toxicity. Recently, super-resolution methods overcame the diffraction barrier to unlock new fields of investigation. Single molecule approaches prove especially useful for three-dimensional nanoscale imaging, and allow couplings between different detection modalities. Still, their use is hindered by the complexity of the…

Advisors/Committee Members: Lévêque-Fort, Sandrine (thesis director).

Subjects/Keywords: Optique; Microscopie; Fluorescence; Super-résolution; Biologie; Optics; Microscopy; Fluorescence; Super-resolution; Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Cabriel, C. (2019). Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie. (Doctoral Dissertation). Université Paris-Saclay (ComUE). Retrieved from http://www.theses.fr/2019SACLS220

Chicago Manual of Style (16^th Edition):

Cabriel, Clément. “Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie.” 2019. Doctoral Dissertation, Université Paris-Saclay (ComUE). Accessed January 20, 2021. http://www.theses.fr/2019SACLS220.

MLA Handbook (7^th Edition):

Cabriel, Clément. “Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie.” 2019. Web. 20 Jan 2021.

Vancouver:

Cabriel C. Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie. [Internet] [Doctoral dissertation]. Université Paris-Saclay (ComUE); 2019. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2019SACLS220.

Council of Science Editors:

Cabriel C. Three-dimensional and multicolour approaches in super-resolution fluorescence microscopy for biology : Approches tri-dimensionnelles et multicolores en microscopie de fluorescence super-résolue pour la biologie. [Doctoral Dissertation]. Université Paris-Saclay (ComUE); 2019. Available from: http://www.theses.fr/2019SACLS220

14. Jouchet, Pierre. Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules.

Degree: Docteur es, Optique et photonique, 2020, université Paris-Saclay

URL: http://www.theses.fr/2020UPASP005

►

L’imagerie tridimensionnelle par localisation de molécules uniques (SMLM) permet d’obtenir des résolutions de quelques dizaines de nanomètres mais présentent encore certaines limitations liées notamment à… (more)

▼

L’imagerie tridimensionnelle par localisation de molécules uniques (SMLM) permet d’obtenir des résolutions de quelques dizaines de nanomètres mais présentent encore certaines limitations liées notamment à une précision axiale non uniforme et une profondeur d’observation souvent limitée au premier micron de l’échantillon. Nous proposons ici une nouvelle approche de localisation de molécules uniques, appelée ModLoc, qui repose sur la modulation et la démodulation du signal de fluorescence des molécules uniques via l’utilisation d’une excitation structurée et modulée temporellement. Dans un premier temps, nous exposons les fondamentaux de l’imagerie SMLM et les limites actuelles de ce domaine. Les subtilités de ce nouveau principe de localisation sont ensuite détaillées et montrent un gain de précision théorique d’un facteur 3. Des études du caractère temporel aléatoire de l’émission des sondes fluorescentes en imagerie SMLM révèlent la nécessité d’intégrer des solutions optiques rapides (proche du kHz). La validation expérimentale du gain en précision accessible est démontrée grâce à la mise en place de deux dispositifs optiques. Nous choisissons d’appliquer ce principe afin d’améliorer la précision de localisation axiale des molécules fluorescentes. Les résultats obtenus mettent en évidence une précision de localisation uniforme de 7.5 nm et jusqu’à 7 microns en profondeur sur des échantillons de calibrations et des échantillons biologiques. La robustesse de la méthode pour l’imagerie SMLM en profondeur est également démontrée grâce notamment à des acquisitions effectuées à 30 µm de profondeur dans des milieux aberrants. Différentes pistes d’amélioration du dispositif actuel ainsi que l’extension de cette approche de localisation modulée à l’observation d’autres grandeurs telle que le temps de vie et l’orientation des molécules fluorescentes sont proposées.

Three-dimensional imaging by localization of single molecules (SMLM) makes it possible to obtain resolutions of a few tens of nanometers, but still has certain limitations related in particular to non-uniform axial precision and a depth of observation often limited to the first micron of the sample. We propose here a new approach to single molecule localization, called ModLoc, which is based on the modulation and demodulation of the fluorescence signal of single molecules through the use of structured and time-modulated excitation. First, we present the fundamentals of SMLM imaging and the current limitations of this field. The subtleties of this new localization principle are then detailed and show a theoretical gain in precision by a factor of 3. Temporal emission studies of fluorescent probes in SMLM imaging reveal the need to integrate fast optical solutions (close to kHz). Experimental validation of the precision gain is demonstrated by the implementation of two optical devices. We choose to apply this principle in order to improve the accuracy of axial localization of fluorescent molecules. The results obtained show a uniform localization precision of 7.5…

Advisors/Committee Members: Lévêque-Fort, Sandrine (thesis director), Poüs, Christian (thesis director).

Subjects/Keywords: Microscopie; Fluorescence; Super-résolution; Localisation; Molécule unique; Microscopy; Fluorescence; Super-resolution; Localization; Single molecule

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APA (6^th Edition):

Jouchet, P. (2020). Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules. (Doctoral Dissertation). université Paris-Saclay. Retrieved from http://www.theses.fr/2020UPASP005

Chicago Manual of Style (16^th Edition):

Jouchet, Pierre. “Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules.” 2020. Doctoral Dissertation, université Paris-Saclay. Accessed January 20, 2021. http://www.theses.fr/2020UPASP005.

MLA Handbook (7^th Edition):

Jouchet, Pierre. “Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules.” 2020. Web. 20 Jan 2021.

Vancouver:

Jouchet P. Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules. [Internet] [Doctoral dissertation]. université Paris-Saclay; 2020. [cited 2021 Jan 20]. Available from: http://www.theses.fr/2020UPASP005.

Council of Science Editors:

Jouchet P. Microscopie super-résolue tridimensionnelle par modulation du signal de fluorescence de molécules uniques : Three-dimensional super-resolved microscopy by modulation of the fluorescence signal of single molecules. [Doctoral Dissertation]. université Paris-Saclay; 2020. Available from: http://www.theses.fr/2020UPASP005

15. Abdul Rehman, Sohaib. Super-resolution microscopy : novel developments and optimisations.

Degree: PhD, 2019, University of Cambridge

URL: https://doi.org/10.17863/CAM.43503 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787773

► 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… (more)

Subjects/Keywords: Fluorescence Microscopy; Super-resolution Microscopy; Localisation Microscopy; Three-dimensional Microscopy; Lightfield Microscopy; Single Molecule Tracking; Clustering; Notch Pathway; Chromatin Architecture; Optics

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APA (6^th Edition):

Abdul Rehman, S. (2019). Super-resolution microscopy : novel developments and optimisations. (Doctoral Dissertation). University of Cambridge. Retrieved from https://doi.org/10.17863/CAM.43503 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787773

Chicago Manual of Style (16^th Edition):

Abdul Rehman, Sohaib. “Super-resolution microscopy : novel developments and optimisations.” 2019. Doctoral Dissertation, University of Cambridge. Accessed January 20, 2021. https://doi.org/10.17863/CAM.43503 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787773.

MLA Handbook (7^th Edition):

Abdul Rehman, Sohaib. “Super-resolution microscopy : novel developments and optimisations.” 2019. Web. 20 Jan 2021.

Vancouver:

Abdul Rehman S. Super-resolution microscopy : novel developments and optimisations. [Internet] [Doctoral dissertation]. University of Cambridge; 2019. [cited 2021 Jan 20]. Available from: https://doi.org/10.17863/CAM.43503 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787773.

Council of Science Editors:

Abdul Rehman S. Super-resolution microscopy : novel developments and optimisations. [Doctoral Dissertation]. University of Cambridge; 2019. Available from: https://doi.org/10.17863/CAM.43503 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.787773

University of Cambridge

16. Abdul Rehman, Sohaib. Super-resolution Microscopy: Novel Developments and Optimisations.

Degree: PhD, 2019, University of Cambridge

URL: https://www.repository.cam.ac.uk/handle/1810/296453

► 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… (more)

Subjects/Keywords: Fluorescence Microscopy; Super-resolution Microscopy; Localisation Microscopy; Three-dimensional Microscopy; Lightfield Microscopy; Single Molecule Tracking; Clustering; Notch Pathway; Chromatin Architecture; Optics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Abdul Rehman, S. (2019). Super-resolution Microscopy: Novel Developments and Optimisations. (Doctoral Dissertation). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/296453

Chicago Manual of Style (16^th Edition):

Abdul Rehman, Sohaib. “Super-resolution Microscopy: Novel Developments and Optimisations.” 2019. Doctoral Dissertation, University of Cambridge. Accessed January 20, 2021. https://www.repository.cam.ac.uk/handle/1810/296453.

MLA Handbook (7^th Edition):

Abdul Rehman, Sohaib. “Super-resolution Microscopy: Novel Developments and Optimisations.” 2019. Web. 20 Jan 2021.

Vancouver:

Abdul Rehman S. Super-resolution Microscopy: Novel Developments and Optimisations. [Internet] [Doctoral dissertation]. University of Cambridge; 2019. [cited 2021 Jan 20]. Available from: https://www.repository.cam.ac.uk/handle/1810/296453.

Council of Science Editors:

Abdul Rehman S. Super-resolution Microscopy: Novel Developments and Optimisations. [Doctoral Dissertation]. University of Cambridge; 2019. Available from: https://www.repository.cam.ac.uk/handle/1810/296453

University of California – Santa Cruz

17. Kissel, Matthew. Design and Function of a Structured Illumination System For Use In An AO Microscope.

Degree: Electrical Engineering, 2014, University of California – Santa Cruz

URL: http://www.escholarship.org/uc/item/1xt7m2q3

► The design and function of a Strucutred Illumination system for use as part of an Adaptive Optics Structured Illumination Microscope (AOSIM) is presented. After successful… (more)

Subjects/Keywords: Optics; Electrical engineering; microscopy; Structured Illumination; Super-resolution

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APA (6^th Edition):

Kissel, M. (2014). Design and Function of a Structured Illumination System For Use In An AO Microscope. (Thesis). University of California – Santa Cruz. Retrieved from http://www.escholarship.org/uc/item/1xt7m2q3

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Kissel, Matthew. “Design and Function of a Structured Illumination System For Use In An AO Microscope.” 2014. Thesis, University of California – Santa Cruz. Accessed January 20, 2021. http://www.escholarship.org/uc/item/1xt7m2q3.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Kissel, Matthew. “Design and Function of a Structured Illumination System For Use In An AO Microscope.” 2014. Web. 20 Jan 2021.

Vancouver:

Kissel M. Design and Function of a Structured Illumination System For Use In An AO Microscope. [Internet] [Thesis]. University of California – Santa Cruz; 2014. [cited 2021 Jan 20]. Available from: http://www.escholarship.org/uc/item/1xt7m2q3.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kissel M. Design and Function of a Structured Illumination System For Use In An AO Microscope. [Thesis]. University of California – Santa Cruz; 2014. Available from: http://www.escholarship.org/uc/item/1xt7m2q3

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Cornell University

18. Shelby, Sarah. Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy.

Degree: PhD, Biophysics, 2015, Cornell University

URL: http://hdl.handle.net/1813/39405

► Allergic signaling is initiated on the plasma membrane of mast cells through cross-linking of immunoglobulin E (IgE) bound to its receptor, Fc[epsilon]RI, by multivalent antigen.… (more)

▼ Allergic signaling is initiated on the plasma membrane of mast cells through cross-linking of immunoglobulin E (IgE) bound to its receptor, Fc[epsilon]RI, by multivalent antigen. Signaling proceeds through controlled spatial assembly of Fc[epsilon]RI receptors with interaction partners in a process that gives rise to specific signaling outcomes. Antigenstimulated redistribution of signaling molecules has proven difficult to characterize by fluorescence imaging due to the small length scales over which this redistribution occurs. We employ super-resolution fluorescence localization microscopy to measure antigen-stimulated changes in the nanoscale organization and mobility of Fc[epsilon]RI, as well as its spatial association with its signaling partner Lyn kinase, with the goal of understanding the physical mechanisms by which spatial redistribution of signaling molecules on the membrane causes the initiation of the signaling response. Using super-resolution imaging, we record receptor organization and dynamics on live mast cells undergoing antigen-mediated signaling, allowing us to measure nanoscale clustering and diffusion of Fc[epsilon]RI simultaneously. Through comparison of cross-linking-induced changes in these properties as a function of time, we are able to resolve two distinct temporal phases of receptor clustering and immobilization. We correlate the time-dependence of the distinct phases with a functional signaling iii response, Ca2+ mobilization, to assess the relevance of each phase to the onset of signaling. We also use super-resolution imaging to measure interactions of Fc[epsilon]RI with Lyn. Because of the improved resolution of our imaging technique, we detect coupling of Lyn to cross-linked IgE-Fc[epsilon]RI that occurs at early stages after antigen stimulation and is associated with the initiation of the stimulated response. Lyn association with Fc[epsilon]RI is assessed through direct imaging, and we can measure the average physical properties of Lyn/Fc[epsilon]RI co-clusters. Through this quantitative approach, we examine mechanisms of regulation of Lyn co-redistribution with IgE-Fc[epsilon]RI. In particular we show that the actin cytoskeleton negatively regulates Fc[epsilon]RI signaling by reducing the local accumulation of Lyn with Fc[epsilon]RI clusters during the onset of the response. The spatial resolution afforded by this technique makes it an effective tool for investigating interactions that give rise to the changes in the organization or mobility of signaling molecules during the initiation of signaling, and how these changes translate into cellular functions. iv Advisors/Committee Members: Baird, Barbara Ann (chair), Zipfel, Warren R. (coChair), Holowka, David Allan (committee member), Chen, Peng (committee member).

Subjects/Keywords: super-resolution localization microscopy; IgE receptor; membrane signaling

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Shelby, S. (2015). Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy. (Doctoral Dissertation). Cornell University. Retrieved from http://hdl.handle.net/1813/39405

Chicago Manual of Style (16^th Edition):

Shelby, Sarah. “Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy.” 2015. Doctoral Dissertation, Cornell University. Accessed January 20, 2021. http://hdl.handle.net/1813/39405.

MLA Handbook (7^th Edition):

Shelby, Sarah. “Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy.” 2015. Web. 20 Jan 2021.

Vancouver:

Shelby S. Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy. [Internet] [Doctoral dissertation]. Cornell University; 2015. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1813/39405.

Council of Science Editors:

Shelby S. Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy. [Doctoral Dissertation]. Cornell University; 2015. Available from: http://hdl.handle.net/1813/39405

University of Michigan

19. Rowland, David. Improving the Scope and Quality of Single-Molecule Data Analysis.

Degree: PhD, Biophysics, 2016, University of Michigan

URL: http://hdl.handle.net/2027.42/137112

► In this dissertation, I extend the scope and quality of the information that may be gained from a single-molecule imaging data set. First, I investigate… (more)

▼ In this dissertation, I extend the scope and quality of the information that may be gained from a single-molecule imaging data set. First, I investigate a more analytically accurate point spread fitting function for use in determining not only the location of fluorescent molecules but also its in-frame displacement. This contrasts with previous work that determined the displacement through an empirically derived calibration function. Some improvements are shown to be possible by using the new sum of error functions (SErf) fitting function instead of the previously used asymmetric Gaussian. Secondly, I analyze the extremely quickly diffusing molecules inside bacteria. These diffusing molecules are found to be so blurred by in-frame motion so as to prohibit accurate localization and thus preclude the subsequent use of single particle tracking for determining the molecules’ diffusive properties. Instead I implement for the first time in bacteria spatio-temporal image correlation spectroscopy (STICS), which does not suffer from in-frame blur in the same way as SPT. I did, however, find two biases inherent to STICS and describe their causes to be experimental deviation from the expected diffusive step size distributions due to in-frame motion and tight confinement. Finally I consider the multi-step fitting process commonly used to determine the mean squared displacement (MSD) of diffusing molecules via the cumulative probability distribution (CPD). I find that by combining the CPD and MSD fits into a single, multi-domain fit, the number of free parameters in the fitting procedure can be reduced from dozens to fewer than 10. This reduction in the fitting degrees of freedom is due to combining redundant parameters, and both improved the precision of diffusion coefficient estimation as well as the likelihood that the fitting procedure produced physically relevant results. These three areas of focus expand the scope of single-molecule super-resolution data analysis by simplifying a method of determining instantaneous displacements of point light sources, and improve the quality of both spatiotemporal image correlation spectroscopy in bacteria and single-particle tracking analysis via cumulative probability distributions of squared step sizes. Advisors/Committee Members: Biteen, Julie Suzanne (committee member), Fessler, Jeffrey A (committee member), Meiners, Jens-Christian D (committee member), Walter, Nils G (committee member).

Subjects/Keywords: Single-molecule super-resolution microscopy analysis methods; Physics; Science

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rowland, D. (2016). Improving the Scope and Quality of Single-Molecule Data Analysis. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/137112

Chicago Manual of Style (16^th Edition):

Rowland, David. “Improving the Scope and Quality of Single-Molecule Data Analysis.” 2016. Doctoral Dissertation, University of Michigan. Accessed January 20, 2021. http://hdl.handle.net/2027.42/137112.

MLA Handbook (7^th Edition):

Rowland, David. “Improving the Scope and Quality of Single-Molecule Data Analysis.” 2016. Web. 20 Jan 2021.

Vancouver:

Rowland D. Improving the Scope and Quality of Single-Molecule Data Analysis. [Internet] [Doctoral dissertation]. University of Michigan; 2016. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2027.42/137112.

Council of Science Editors:

Rowland D. Improving the Scope and Quality of Single-Molecule Data Analysis. [Doctoral Dissertation]. University of Michigan; 2016. Available from: http://hdl.handle.net/2027.42/137112

20. Liao, Yi. Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy.

Degree: PhD, Chemistry, 2015, University of Michigan

URL: http://hdl.handle.net/2027.42/113498

► The error-free progression of DNA replication is essential for all organisms. Approximately 80 known human diseases are caused by malfunction in DNA replication, and deficiencies… (more)

▼ The error-free progression of DNA replication is essential for all organisms. Approximately 80 known human diseases are caused by malfunction in DNA replication, and deficiencies in DNA repair mechanisms can also lead to severe consequences such as increased antibiotic resistance in bacteria and cancers in humans. A better understanding of DNA replication and repair requires knowledge of the key players along relevant pathways at the molecular level and in the cellular context. This characterization calls for a technique with superior sensitivity, accuracy and biocompatibility. In this thesis, we integrate single-molecule super-resolution microscopy and single-particle tracking with genetic and genomic approaches to study two proteins that play a pivotal role in maintaining genomic integrity: MutS and PolC. From prokaryotes to human cells, homologs of the highly conserved mismatch repair (MMR) protein MutS recognize mispaired nucleotides and recruit the proteins responsible for downstream repair. Although the structure and function of MutS have been extensively characterized in biochemical isolation, it remains unclear how MutS efficiently identifies, among millions of correctly paired bases, a single mismatch in the complex and crowded cellular environment. To obtain mechanistic insights into MMR initiation from an in vivo perspective, we applied super-resolution imaging in live Bacillus subtilis cells to follow the motion of single MutS proteins in real time, and we monitored how MutS behavior is affected by sequentially blocking critical steps along the MMR pathway. Our results demonstrate an intimate and dynamic coupling between MutS and the replisome which stages MutS to sites of DNA replication, allowing MutS to scan newly synthesized DNA in anticipation of errors largely free of obstacles. We then turn our focus to DNA replication itself. Specifically, we focused on PolC, one of the two essential DNA polymerases in B. subtilis. Based on photobleaching-assisted microscopy and three-dimensional super-resolution microscopy, we quantified the stoichiometry, intracellular locations and dynamics of PolC. Finally, we extended the application of super-resolution microscopy to the field of renewable energy by tracking single molecules and visualizing guest-host interactions in microporous coordination polymers (MCPs). Advisors/Committee Members: Biteen, Julie Suzanne (committee member), Ogilvie, Jennifer P. (committee member), Veatch, Sarah (committee member), Simmons, Lyle A. (committee member), Chen, Zhan (committee member).

Subjects/Keywords: Super-resolution microscopy; Single-molecule imaging; DNA mismatch repair; Chemistry; Science

11 more images…

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APA (6^th Edition):

Liao, Y. (2015). Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/113498

Chicago Manual of Style (16^th Edition):

Liao, Yi. “Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy.” 2015. Doctoral Dissertation, University of Michigan. Accessed January 20, 2021. http://hdl.handle.net/2027.42/113498.

MLA Handbook (7^th Edition):

Liao, Yi. “Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy.” 2015. Web. 20 Jan 2021.

Vancouver:

Liao Y. Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy. [Internet] [Doctoral dissertation]. University of Michigan; 2015. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2027.42/113498.

Council of Science Editors:

Liao Y. Single-Molecule Localization, Dynamics and Interactions of DNA Replication and Repair Proteins Revealed by Live-Cell Super-Resolution Microscopy. [Doctoral Dissertation]. University of Michigan; 2015. Available from: http://hdl.handle.net/2027.42/113498

University of Michigan

21. Rowland, David. Improving the Scope and Quality of Single-Molecule Data Analysis.

Degree: PhD, Biophysics, 2016, University of Michigan

URL: http://hdl.handle.net/2027.42/135862

► In this dissertation, I extend the scope and quality of the information that may be gained from a single-molecule imaging data set. First, I investigate… (more)

▼ In this dissertation, I extend the scope and quality of the information that may be gained from a single-molecule imaging data set. First, I investigate a more analytically accurate point spread fitting function for use in determining not only the location of fluorescent molecules but also its in-frame displacement. This contrasts with previous work that determined the displacement through an empirically derived calibration function. Some improvements are shown to be possible by using the new sum of error functions (SErf) fitting function instead of the previously used asymmetric Gaussian. Secondly, I analyze the extremely quickly diffusing molecules inside bacteria. These diffusing molecules are found to be so blurred by in-frame motion so as to prohibit accurate localization and thus preclude the subsequent use of single particle tracking for determining the molecules’ diffusive properties. Instead I implement for the first time in bacteria spatio-temporal image correlation spectroscopy (STICS), which does not suffer from in-frame blur in the same way as SPT. I did, however, find two biases inherent to STICS and describe their causes to be experimental deviation from the expected diffusive step size distributions due to in-frame motion and tight confinement. Finally I consider the multi-step fitting process commonly used to determine the mean squared displacement (MSD) of diffusing molecules via the cumulative probability distribution (CPD). I find that by combining the CPD and MSD fits into a single, multi-domain fit, the number of free parameters in the fitting procedure can be reduced from dozens to fewer than 10. This reduction in the fitting degrees of freedom is due to combining redundant parameters, and both improved the precision of diffusion coefficient estimation as well as the likelihood that the fitting procedure produced physically relevant results. These three areas of focus expand the scope of single-molecule super-resolution data analysis by simplifying a method of determining instantaneous displacements of point light sources, and improve the quality of both spatiotemporal image correlation spectroscopy in bacteria and single-particle tracking analysis via cumulative probability distributions of squared step sizes. Advisors/Committee Members: Biteen, Julie Suzanne (committee member), Fessler, Jeffrey A (committee member), Meiners, Jens-Christian D (committee member), Walter, Nils G (committee member).

Subjects/Keywords: Single-molecule super-resolution microscopy analysis methods; Physics; Science

Record Details Similar Records Cite « Share

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Rowland, D. (2016). Improving the Scope and Quality of Single-Molecule Data Analysis. (Doctoral Dissertation). University of Michigan. Retrieved from http://hdl.handle.net/2027.42/135862

Chicago Manual of Style (16^th Edition):

Rowland, David. “Improving the Scope and Quality of Single-Molecule Data Analysis.” 2016. Doctoral Dissertation, University of Michigan. Accessed January 20, 2021. http://hdl.handle.net/2027.42/135862.

MLA Handbook (7^th Edition):

Rowland, David. “Improving the Scope and Quality of Single-Molecule Data Analysis.” 2016. Web. 20 Jan 2021.

Vancouver:

Rowland D. Improving the Scope and Quality of Single-Molecule Data Analysis. [Internet] [Doctoral dissertation]. University of Michigan; 2016. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/2027.42/135862.

Council of Science Editors:

Rowland D. Improving the Scope and Quality of Single-Molecule Data Analysis. [Doctoral Dissertation]. University of Michigan; 2016. Available from: http://hdl.handle.net/2027.42/135862

University of Toronto

22. Hadipour-Lakmehsari, Sina. Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease.

Degree: 2018, University of Toronto

URL: http://hdl.handle.net/1807/97649

►

The nucleoplasmic reticulum (NR) and sarcoplasmic reticulum (SR) are critical intracellular subdomains within the cardiomyocyte. Despite their importance, there are large gaps of knowledge regarding… (more)

Subjects/Keywords: Cardiac; Nucleoplasmic reticulum; Pressure-overload cardiac hypertrophy; Super-resolution microscopy; 0379

Record Details Similar Records Cite « Share

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hadipour-Lakmehsari, S. (2018). Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/97649

Chicago Manual of Style (16^th Edition):

Hadipour-Lakmehsari, Sina. “Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease.” 2018. Masters Thesis, University of Toronto. Accessed January 20, 2021. http://hdl.handle.net/1807/97649.

MLA Handbook (7^th Edition):

Hadipour-Lakmehsari, Sina. “Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease.” 2018. Web. 20 Jan 2021.

Vancouver:

Hadipour-Lakmehsari S. Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease. [Internet] [Masters thesis]. University of Toronto; 2018. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1807/97649.

Council of Science Editors:

Hadipour-Lakmehsari S. Analysis of Specialized Subcellular Regions in Cardiac Myocytes in Health and Disease. [Masters Thesis]. University of Toronto; 2018. Available from: http://hdl.handle.net/1807/97649

University of Manchester

23. Srpan, Katja. Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16.

Degree: 2017, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312497

► Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing… (more)

▼ Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. The strength of perforin secretion is also translated to killing of Daudi target cells, expressing different ligands. When Daudi, opsonised with rituximab is the first target NK cell encounters, the sequential killing of another opsonised rituximab or Daudi, expressing MICA will not be affected. But, when Daudi-MICA is met first, the consecutive killing of Daudi-MICA as well as Daudi-rituximab will be impaired. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both, CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation through NKG2D. Interestingly, however, we also identified the beneficial role of CD16 shedding for NK cell serial killing. NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Moreover, the inhibition of CD16 shedding also prevents the NK cell detachment from rituximab opsonised Daudi cells. Thus, the shedding of the receptor can serve to augment NK cell motility to move between target cells. Efficient NK cell detachment also correlated with their increased survival. Finally, we report that CD16 is constitutively organised in small, dense nanoclusters and that the ligation with rituximab does not affect their spatial distribution. Despite the shedding of the receptor, leading to less protein molecules at the surface, the area of these clusters remains the same. Together these data suggest that CD16 shedding hinders NK cell cytotoxicity against opsonised targets, but promotes their movements between different targets. Thus, receptor shedding is important for efficient NK cell serial killing. Manipulation of CD16 shedding, perhaps by boosting its recovery, might therefore represent an important target for NK cell-based therapies including treatments with therapeutic antibodies. Advisors/Committee Members: TRAVIS, MARK MA, Davis, Daniel, Travis, Mark.

Subjects/Keywords: NK cell; CD16; serial killing; ADCC; cell detachment; super-resolution microscopy

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Srpan, K. (2017). Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312497

Chicago Manual of Style (16^th Edition):

Srpan, Katja. “Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16.” 2017. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312497.

MLA Handbook (7^th Edition):

Srpan, Katja. “Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16.” 2017. Web. 20 Jan 2021.

Vancouver:

Srpan K. Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16. [Internet] [Doctoral dissertation]. University of Manchester; 2017. [cited 2021 Jan 20]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312497.

Council of Science Editors:

Srpan K. Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16. [Doctoral Dissertation]. University of Manchester; 2017. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312497

University of Washington

24. Chozinski, Tyler Joseph. Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems.

Degree: PhD, 2018, University of Washington

URL: http://hdl.handle.net/1773/42234

► Super-resolution fluorescence microscopy enables researchers to directly observe details of biological systems on the nanoscale. However, current methods are often costly, require considerable skill, and… (more)

Subjects/Keywords: expansion; fluorescence; glomerulus; kidney; microscopy; Super-resolution; Chemistry; Chemistry

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APA (6^th Edition):

Chozinski, T. J. (2018). Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/42234

Chicago Manual of Style (16^th Edition):

Chozinski, Tyler Joseph. “Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems.” 2018. Doctoral Dissertation, University of Washington. Accessed January 20, 2021. http://hdl.handle.net/1773/42234.

MLA Handbook (7^th Edition):

Chozinski, Tyler Joseph. “Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems.” 2018. Web. 20 Jan 2021.

Vancouver:

Chozinski TJ. Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems. [Internet] [Doctoral dissertation]. University of Washington; 2018. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1773/42234.

Council of Science Editors:

Chozinski TJ. Expansion Microscopy for the Interrogation of Nanoscale Features in Complex Biological Systems. [Doctoral Dissertation]. University of Washington; 2018. Available from: http://hdl.handle.net/1773/42234

University of Washington

25. Howard, Marco d. A New Approach to 3-Dimensional Super-Resolution Microscopy.

Degree: PhD, 2020, University of Washington

URL: http://hdl.handle.net/1773/45139

► Super-Resolution microscopy has transformed our ability to image biological specimens at the nanoscale with high contrast and molecular specificity. However, acquiring 3-dimensional images over large… (more)

Subjects/Keywords: DNA-Barcode; Imaging; Immunofluorescence; Microscopy; Nanoscopy; Super-Resolution; Bioengineering; Chemistry

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APA (6^th Edition):

Howard, M. d. (2020). A New Approach to 3-Dimensional Super-Resolution Microscopy. (Doctoral Dissertation). University of Washington. Retrieved from http://hdl.handle.net/1773/45139

Chicago Manual of Style (16^th Edition):

Howard, Marco d. “A New Approach to 3-Dimensional Super-Resolution Microscopy.” 2020. Doctoral Dissertation, University of Washington. Accessed January 20, 2021. http://hdl.handle.net/1773/45139.

MLA Handbook (7^th Edition):

Howard, Marco d. “A New Approach to 3-Dimensional Super-Resolution Microscopy.” 2020. Web. 20 Jan 2021.

Vancouver:

Howard Md. A New Approach to 3-Dimensional Super-Resolution Microscopy. [Internet] [Doctoral dissertation]. University of Washington; 2020. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1773/45139.

Council of Science Editors:

Howard Md. A New Approach to 3-Dimensional Super-Resolution Microscopy. [Doctoral Dissertation]. University of Washington; 2020. Available from: http://hdl.handle.net/1773/45139

Rice University

26. Baiyasi, Rashad. Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters.

Degree: MS, Engineering, 2018, Rice University

URL: http://hdl.handle.net/1911/105721

► Plasmonic nanostructures offer a wealth of novel optical properties but optimization of their structure-function relationship with superlocalization techniques is hindered by the modification of point… (more)

Subjects/Keywords: plasmonics; nanowires; superlocalization; Hermite-Gaussian; super-resolution microscopy

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APA (6^th Edition):

Baiyasi, R. (2018). Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters. (Masters Thesis). Rice University. Retrieved from http://hdl.handle.net/1911/105721

Chicago Manual of Style (16^th Edition):

Baiyasi, Rashad. “Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters.” 2018. Masters Thesis, Rice University. Accessed January 20, 2021. http://hdl.handle.net/1911/105721.

MLA Handbook (7^th Edition):

Baiyasi, Rashad. “Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters.” 2018. Web. 20 Jan 2021.

Vancouver:

Baiyasi R. Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters. [Internet] [Masters thesis]. Rice University; 2018. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1911/105721.

Council of Science Editors:

Baiyasi R. Plasmonic modulation of fluorescence point spread functions reveals underlying properties of quantum emitters. [Masters Thesis]. Rice University; 2018. Available from: http://hdl.handle.net/1911/105721

University of Notre Dame

27. Genevieve Vigil. Label-Free and Super-Resolution Multiphoton Microscopy</h1>.

Degree: Electrical Engineering, 2017, University of Notre Dame

URL: https://curate.nd.edu/show/wm117m04263

► Multiphoton Microscopy (MPM) has provided the biomedical imaging field a methodology to perform high resolution, 3D, fluorescent imaging deeper and with less photodamage than… (more)

▼ Multiphoton Microscopy (MPM) has provided the biomedical imaging field a methodology to perform high resolution, 3D, fluorescent imaging deeper and with less photodamage than other technologies, such as confocal microscopy. These features have proven MPM as an excellent tool for extit{in vivo} studies. However, most MPM studies use external probes or genetically modified animals thereby adding complexity and potentially interfering with the function of the system in question. Further, though MPM is capable of some of the highest resolution imaging at depth, it is bound by diffraction and scattering which limit resolution to sub-cellular levels and penetration depth to mm scales. This work attempts to circumvent the current limitations of MPM in two ways. First, to expand the utility of label free MPM by characterizing intrinsic signals which could be leveraged for meaningful studies of the rare and neglected disease, sickle cell disease (SCD). Secondly, a method is proposed to drive the resolving power and imaging depth of MPM further. Towards these goals, studies of true \ extit{in situ} samples were used without external labels to perform clinically relevant investigations of SCD which could later be useful for extit{in vivo} monitoring in mouse models and therapy testing. Additionally, this work describes a nonlinear microscopy technique which leverages saturation effects for the development of a fluorescence microscopy technology capable of super-resolution imaging in thick, scattering tissue. Imaging of intrinsic two photon excited fluorescence (TPEF) is performed on humanized SCD mouse model splenic tissue. Distinct morphological and spectral features associated with SCD are identified and discussed in terms of diagnostic relevance. Specifically, spectrally unique splenic iron-complex deposits are identified by MPM; this finding is supported by TPEF spectroscopy and object size to standard histopathological methods. Further, iron deposits are found at higher concentrations by all imaging methods employed here including MPM in diseased tissue than in healthy tissue and therefore may provide a useful optical biomarker related to the disease state. These newly characterized biomarkers allow for further investigations of SCD in live animals as a means to gain insight into the mechanisms impacting immune dysregulation and organ malfunction in SCD, which are currently not well understood. Additionally, the photophysical properties of human SCD Hemoglobin (Hb) is characterized by multi-photon microscopy (MPM). The intrinsic TPEF signal associated with extracted hemoglobin was investigated and the solidified SCD variant (HbS) was found to demonstrate broad emission peaking around 510 nm when excited at 800 nm. MPM is used to dynamically induce and image HbS gelling by photolysis of deoxygenated HbS. For comparison, photolysis conditions were applied to a healthy variant of human hemoglobin (HbA) and found to remain in solution not forming fibers. The use of this signal to study the mechanism… Advisors/Committee Members: Scott Howard, Research Director.

Subjects/Keywords: Sickle Cell Disease; Multiphoton; Super-REsolution; Label-Free; Microscopy

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APA (6^th Edition):

Vigil, G. (2017). Label-Free and Super-Resolution Multiphoton Microscopy</h1>. (Thesis). University of Notre Dame. Retrieved from https://curate.nd.edu/show/wm117m04263

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Vigil, Genevieve. “Label-Free and Super-Resolution Multiphoton Microscopy</h1>.” 2017. Thesis, University of Notre Dame. Accessed January 20, 2021. https://curate.nd.edu/show/wm117m04263.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Vigil, Genevieve. “Label-Free and Super-Resolution Multiphoton Microscopy</h1>.” 2017. Web. 20 Jan 2021.

Vancouver:

Vigil G. Label-Free and Super-Resolution Multiphoton Microscopy</h1>. [Internet] [Thesis]. University of Notre Dame; 2017. [cited 2021 Jan 20]. Available from: https://curate.nd.edu/show/wm117m04263.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Vigil G. Label-Free and Super-Resolution Multiphoton Microscopy</h1>. [Thesis]. University of Notre Dame; 2017. Available from: https://curate.nd.edu/show/wm117m04263

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Toronto

28. Driouchi, Amine. Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques.

Degree: PhD, 2019, University of Toronto

URL: http://hdl.handle.net/1807/96175

► CEACAMs are cell surface glycoproteins involved in homo- and heterophilic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. There is growing evidence… (more)

▼ CEACAMs are cell surface glycoproteins involved in homo- and heterophilic intercellular interactions that control cellular growth, differentiation, tumourigenesis, inflammation and infection. There is growing evidence that CEACAM1 isoforms regulate the cellular response to CEACAM1-mediated interactions between various cell types. Reinforcing this point, given its potential as a tumour suppressor and immunomodulator, CEACAM1 is being targeted for clinical development. However, there remain clear challenges owing to the observation that different CEACAM1 isoforms display cell-type specific expression patterns under different physiological conditions. Moreover, the ability of CEACAMs to form dimers and higher-order oligomers, which are thought to impact regulation of intercellular signals, add considerable complexity to our understanding of its functional role(s). Super resolution microscopy has enabled the quantitative characterization of the nanoscale distribution of several membrane proteins, revealing cluster hierarchies at the micro- and nano-scale. However, clustering and self-association are not necessarily the same piece of information and may have very different effects on the cellular response, so mapping the preferential nanoscale distribution of the various self-association states is of particular interest to many. This thesis reports on the development and application of a correlative STORM/homoFRET strategy to study CEACAM1 isoforms and mutants. We have further explored the use of single particle tracking (SPT) and mean square displacement analysis to characterize CEACAM1 dynamics as a function of oligomeric state and location. Building on these datasets, we have applied co-localization and co-occurrence approaches to measure CEACAM1 association to lipid-ordered regions, ezrin and actin. Finally, we present a set of preliminary methods and results that will serve to further the study of membrane protein clustering and self-association and their link to function. Together, this thesis aims to develop a more fulsome perspective on structure, association and dynamics of the CEACAM1 membrane protein while providing methodological approaches to tackle similar questions. Advisors/Committee Members: Yip, Christopher M, Biochemistry.

Subjects/Keywords: CEACAM1; Cell signaling; FRET; Microscopy; Single particle tracking; Super resolution; 0786

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APA (6^th Edition):

Driouchi, A. (2019). Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques. (Doctoral Dissertation). University of Toronto. Retrieved from http://hdl.handle.net/1807/96175

Chicago Manual of Style (16^th Edition):

Driouchi, Amine. “Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques.” 2019. Doctoral Dissertation, University of Toronto. Accessed January 20, 2021. http://hdl.handle.net/1807/96175.

MLA Handbook (7^th Edition):

Driouchi, Amine. “Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques.” 2019. Web. 20 Jan 2021.

Vancouver:

Driouchi A. Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques. [Internet] [Doctoral dissertation]. University of Toronto; 2019. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/1807/96175.

Council of Science Editors:

Driouchi A. Characterization of CEACAM1 Spatial Distribution, Self-association and Dynamics using High-resolution Microscopy Techniques. [Doctoral Dissertation]. University of Toronto; 2019. Available from: http://hdl.handle.net/1807/96175

University of New Mexico

29. Farzam, Farzin. High-Throughput Automated Multi-Target Super-resolution Imaging.

Degree: Physics & Astronomy, 2018, University of New Mexico

URL: https://digitalrepository.unm.edu/phyc_etds/185

► Super-resolution microscopy techniques developed through the past few decades enable us to surpass the classical diffraction limit of light, and thus open new doors… (more)

▼ Super-resolution microscopy techniques developed through the past few decades enable us to surpass the classical diffraction limit of light, and thus open new doors to investigate the formerly inaccessible world of nanometer-sized objects. Most importantly, by using super-resolution microscopy, one can visualize sub-cellular structures in the range of 10 to 200 nm. At this range, we can investigate exciting problems in biology and medicine by visualizing protein-protein interactions and spatiotemporal analysis of structures of interest on the surface or inside cells. These techniques (collectively known as nanoscopy) have a high impact on understanding and solving biological questions. This dissertation starts with a brief and general description of current super-resolution techniques and then moves toward a multi-target super-resolution imaging strategy using sequential imaging that has benefits over conventional multi-color imaging methods. Sequential microscopy takes advantage of the photo-physical properties of the most suitable dye for a particular technique to achieve the optimal and consistent resolution for each of multiple targets of imaging. For example, for dSTORM imaging, this is currently AlexaFluor647. Sequential dSTROM has an advantage for multi-target imaging due to having a single imaging channel which avoids dealing with differential aberration-problems between multiple emission paths unlike other multi-color imaging based methods. We show that sequential imaging method can be facilitated using automated imaging. In this dissertation, a sequential microscope is designed, calibrated, and tested on multiple structures. We show that it can automatically re-find the position of each initially registered cell and can account for sample drift through an entire experiment. The microscope has been used in multiple collaborations with other groups to investigate biological problems of interest. Two labeling strategies that facilitate sequential imaging are described. The first strategy is DNA-strand-displacement , which allows imaging of multiple structures in a controlled and time-efficient binding-unbinding scenario. The second strategy is imaging with the small, actin binding peptide Lifeact. Finally, future directions and suggestions are made about how we can further improve the microscope. In the Appendix I provide a guide on how to use and troubleshoot the microscope, how to measure the efficiency of the microscope, as well as how to fix and label cells for optimal imaging and how to prepare various imaging buffers. Advisors/Committee Members: Dr. Keith A. Lidke, Dr. Diane S. Lidke, Dr. Sudhakar Prasad, Dr. Aaron Neumann.

Subjects/Keywords: Super-Resolution; fluorescence; microscopy; Astrophysics and Astronomy; Physics

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APA (6^th Edition):

Farzam, F. (2018). High-Throughput Automated Multi-Target Super-resolution Imaging. (Doctoral Dissertation). University of New Mexico. Retrieved from https://digitalrepository.unm.edu/phyc_etds/185

Chicago Manual of Style (16^th Edition):

Farzam, Farzin. “High-Throughput Automated Multi-Target Super-resolution Imaging.” 2018. Doctoral Dissertation, University of New Mexico. Accessed January 20, 2021. https://digitalrepository.unm.edu/phyc_etds/185.

MLA Handbook (7^th Edition):

Farzam, Farzin. “High-Throughput Automated Multi-Target Super-resolution Imaging.” 2018. Web. 20 Jan 2021.

Vancouver:

Farzam F. High-Throughput Automated Multi-Target Super-resolution Imaging. [Internet] [Doctoral dissertation]. University of New Mexico; 2018. [cited 2021 Jan 20]. Available from: https://digitalrepository.unm.edu/phyc_etds/185.

Council of Science Editors:

Farzam F. High-Throughput Automated Multi-Target Super-resolution Imaging. [Doctoral Dissertation]. University of New Mexico; 2018. Available from: https://digitalrepository.unm.edu/phyc_etds/185

Duke University

30. Chowdhury, Shwetadwip. Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging .

Degree: 2016, Duke University

URL: http://hdl.handle.net/10161/13362

► Optical microscopy plays a crucial role in the biological sciences for its ability to enable visualization of biological samples at sub-cellular levels. Many imaging… (more)

▼ Optical microscopy plays a crucial role in the biological sciences for its ability to enable visualization of biological samples at sub-cellular levels. Many imaging subdivisions exist under this umbrella of general microscopy, and each are tailored towards specific design, contrast, and visualization constraints. Standard examples that have found widespread use include dark-field, phase-contrast, holographic, and fluorescent microscopies. However, a critical factor that physically limits the optical resolution of general microscopy is diffraction. Unfortunately, this “diffraction-limit” can prevent visualization of significant biologically relevant structures, which in turn can limit biological insights. In response to such a limit, several works have advanced the field of sub-diffraction resolution imaging, which consist of optical imaging techniques that seek to achieve imaging resolutions beyond that which is allowed by the diffraction-limit. This set of techniques can largely be divided into two classes. The first class of sub-diffraction techniques is targeted towards cases where the sample is coherently illuminated and diffracts into the imaging system’s aperture. For such cases, synthetic aperture (SA) is a popular choice and operates by using oblique illuminations to spatiotemporally synthesize a wider frequency support into the image than allowed by the diffraction limit. The second class of sub-diffraction techniques, often referred to as "super-resolution" techniques, typically utilize specialized fluorophores with either photoswitching or depletion capabilities. Photoactivated localization microscopy (PALM) is a super-resolution example that localizes photoswitchable fluorophores to sub-diffraction resolutions per acquisition, before combining into a final super-resolved image. Stimulated emission depletion (STED) is another super-resolution example that spatially modulates its excitation to narrow its optical point-spread-function. Unfortunately, SA and fluorescent super-resolution techniques are generally incompatible for sub-diffraction resolution fluorescent and coherent imaging, respectively – thus, a multimodal sub-diffraction imaging solution compatible with both coherent and fluorescent imaging has remained elusive. In this dissertation, we demonstrate that structured illumination (SI) is a sub-diffraction technique compatible with both diffractive and fluorescent imaging. We first develop the theoretical framework that extends SI to coherent imaging and experimentally demonstrate SI’s capabilities for 2D sub-diffraction resolution imaging of coherently diffractive samples. Sub-diffraction resolution imaging based on scattering intensity and transmission-based quantitative-phase (QP) are shown. In addition, we show extend SI to 3D coherent imaging, and show applications of this towards 3D QP and refractive-index (RI) tomography. Finally, we show multimodal applications of SI that allow sub-diffraction resolution fluorescent and coherent imaging, which has great potential utility for… Advisors/Committee Members: Izatt, Joseph A (advisor).

Subjects/Keywords: Biomedical engineering; microscopy; optics; structured illumination; super-resolution

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APA (6^th Edition):

Chowdhury, S. (2016). Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging . (Thesis). Duke University. Retrieved from http://hdl.handle.net/10161/13362

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Chowdhury, Shwetadwip. “Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging .” 2016. Thesis, Duke University. Accessed January 20, 2021. http://hdl.handle.net/10161/13362.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Chowdhury, Shwetadwip. “Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging .” 2016. Web. 20 Jan 2021.

Vancouver:

Chowdhury S. Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging . [Internet] [Thesis]. Duke University; 2016. [cited 2021 Jan 20]. Available from: http://hdl.handle.net/10161/13362.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Chowdhury S. Technical Developments in Structured Illumination Microscopy for Coherent and Multimodal Fluorescent Sub-Diffraction Resolution Imaging . [Thesis]. Duke University; 2016. Available from: http://hdl.handle.net/10161/13362

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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Open Access Theses and Dissertations