{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/102839"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/102839","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Single quantum dot tracking for quantitative molecular imaging","abstract":"The application of semiconductor nanocrystals known as quantum dots (QDs) to biology in the past few decades has advanced the field of single-molecule biology by allowing for long time-scale tracking of individual biomolecules. However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm.","abstract_html":"The application of semiconductor nanocrystals known as quantum dots (QDs) to biology in the past few decades has advanced the field of single-molecule biology by allowing for long time-scale tracking of individual biomolecules. However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm.","abstract_has_math":false,"creators":["Zahid, Mohammad Usama"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Bioengineering","degree_department":null,"school":null,"contributors":["Smith, Andrew M.","Boppart, Stephen A.","Leal, Cecilia","Selvin, Paul R."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-02-07T20:44:23Z","date_published":"2019-02-07T20:44:23Z","updated_at":"2026-07-22T22:24:42Z","subjects":["Quantum Dots, Single Molecule Imaging, Single Particle Tracking, Quantitative Bioimaging"],"languages":["en"],"rights":["Copyright 2018 Mohammad Usama Zahid"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/102839","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Smith, Andrew M.","Boppart, Stephen A.","Leal, Cecilia","Selvin, Paul R."]},{"key":"dc:creator","label":"Author","values":["Zahid, Mohammad Usama"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-02-07T20:44:23Z","2021-02-08T10:15:11Z","2018-12-05","2018-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Bioengineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Quantum Dots, Single Molecule Imaging, Single Particle Tracking, Quantitative Bioimaging"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2018 Mohammad Usama Zahid"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/102839"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The application of semiconductor nanocrystals known as quantum dots (QDs) to biology in the past few decades has advanced the field of single-molecule biology by allowing for long time-scale tracking of individual biomolecules. However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2020-12-01","The student, Mohammad Zahid, accepted the attached license on 2018-12-05 at 11:21.","The student, Mohammad Zahid, submitted this Dissertation for approval on 2018-12-05 at 11:27.","This Dissertation was approved for publication on 2018-12-05 at 16:26.","DSpace SAF Submission Ingestion Package generated from Vireo submission #13219 on 2019-02-07 at 14:22:40","Made available in DSpace on 2019-02-07T20:44:23Z (GMT). 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However, QD single-molecule imaging studies have almost been exclusively limited to the extracellular space, due to limitations in intracellular delivery techniques and a limited understanding of how nanoparticles behave in the intracellular space. In the first half of this thesis, a new analysis methodology is developed to quantitatively assess the intracellular delivery of QDs. We present a method for using single-molecule imaging and subsequent single-particle tracking (SPT) of QDs delivered to the cytosol of living cells to assess delivery efficiency and uptake mechanisms. In this method, single quantum dot mobility information is used in conjunction with single-molecule brightness measurements to develop novel single-cell metrics of delivery efficiency. These metrics are used to investigate the impact of different nanoparticle surface properties on intracellular delivery and fate. We investigate the delivery of a series of QDs designed with diverse surface properties. This comparison revealed new insights into particle uptake and endosomal escape, as well as the discovery that zwitterionic surfaces are uniquely suited for intracellular mobility. Additionally, this new analysis methodology was validated by established experimental approaches and analysis of simulated single-particle trajectories. The second half of this thesis applies the aforementioned tools towards two applications. The first application is to quantitatively evaluate QD labeling of intracellular proteins in live cells. This was achieved by delivering QDs conjugated to biorthogonal functional groups to label a target protein and developing a new colocalization-based metric to quantify the degree of protein target labeling. We present evidence of protein target labeling by using this single-trajectory level colocalization metric in combination with nanoparticle mobility measurements. The second application is toward accurate measurement of hydrodynamic size of colloids with dimensions smaller than 100 nanometers (nm). We develop a new method using widefield fluorescence microscopy and SPT to measure nanoparticle size and demonstrate accurate single-molecule size measurements of a homogeneous nanoparticle population with hydrodynamic diameter of approximately 20 nm.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2020-12-01","The student, Mohammad Zahid, accepted the attached license on 2018-12-05 at 11:21.","The student, Mohammad Zahid, submitted this Dissertation for approval on 2018-12-05 at 11:27.","This Dissertation was approved for publication on 2018-12-05 at 16:26.","DSpace SAF Submission Ingestion Package generated from Vireo submission #13219 on 2019-02-07 at 14:22:40","Made available in DSpace on 2019-02-07T20:44:23Z (GMT). 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