Superresolution Nonlinear Structured Illumination Microscopy By Stimulated Emission Depletion

Persistent Link:
http://hdl.handle.net/10150/338899
Title:
Superresolution Nonlinear Structured Illumination Microscopy By Stimulated Emission Depletion
Author:
Zhang, Han
Issue Date:
2014
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy (SIM) has been demonstrated a fast and full field superresolution imaging tool, such as Saturated-SIM and Photoswitching-SIM. In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion (STED) field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full field imaging over 1 x 10 micron square at the speed of 2s/frame with 4-fold improved resolution. Our STED-SIM technique has been applied on biological samples and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance (SPR), to nonlinear STED-SIM has been made and around 8 time enhancement on STED quenching effect was achieved. Based on this enhancement on STED, 1D SPR enhanced STED-SIM was built and 50 nm resolution of fluorescence beads sample was achieved. Algorithm improvement is required to achieve full field superresolution imaging with SPR enhanced STED-SIM. The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two photon Bessel beam light-sheet microscopy and nonlinear SIM, 3D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebrafish. Two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity.
Type:
text; Electronic Dissertation
Keywords:
light-sheet microscopy; Nonlinear; Stimulated emission depletion; Structured illumination; Superresolution; Fluorescence microscopy; Optical Sciences
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Optical Sciences
Degree Grantor:
University of Arizona
Advisor:
Peng, Leilei

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleSuperresolution Nonlinear Structured Illumination Microscopy By Stimulated Emission Depletionen_US
dc.creatorZhang, Hanen_US
dc.contributor.authorZhang, Hanen_US
dc.date.issued2014-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy (SIM) has been demonstrated a fast and full field superresolution imaging tool, such as Saturated-SIM and Photoswitching-SIM. In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion (STED) field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full field imaging over 1 x 10 micron square at the speed of 2s/frame with 4-fold improved resolution. Our STED-SIM technique has been applied on biological samples and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance (SPR), to nonlinear STED-SIM has been made and around 8 time enhancement on STED quenching effect was achieved. Based on this enhancement on STED, 1D SPR enhanced STED-SIM was built and 50 nm resolution of fluorescence beads sample was achieved. Algorithm improvement is required to achieve full field superresolution imaging with SPR enhanced STED-SIM. The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two photon Bessel beam light-sheet microscopy and nonlinear SIM, 3D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebrafish. Two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity.en_US
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectlight-sheet microscopyen_US
dc.subjectNonlinearen_US
dc.subjectStimulated emission depletionen_US
dc.subjectStructured illuminationen_US
dc.subjectSuperresolutionen_US
dc.subjectFluorescence microscopyen_US
dc.subjectOptical Sciencesen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineOptical Sciencesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorPeng, Leileien_US
dc.contributor.committeememberPeng, Leileien_US
dc.contributor.committeememberMilster, Tom D.en_US
dc.contributor.committeememberLiang, Rongguangen_US
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