{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/95526"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/95526","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Intracellular labeling of live cells via transient membrane permeabilization for fluorescence and super resolution microscopy","abstract":"Fluorescence imaging of intracellular proteins is often achieved by using transfection-induced expression of fluorescent protein. This can potentially impose artifacts such as loss of function or over-expression of target proteins. Direct labeling of the intracellular protein is an alternative to transfection, but is largely limited by permeability of the fluorescent probes. Here, we have developed a high-throughput technique for labeling intracellular proteins of living cells. The technique makes use of Streptolysin O (SLO), a bacterial enzyme that permeabilizes cells to DNA, RNA, proteins. We show that SLO can be used to deliver a variety of fluorescent probes; ranging from organic dyes (<1 kDa in size) to fluorescent immunoglobulin antibody (~150 kDa) for specific labeling of intracellular proteins. We demonstrate in numerous ways that after permeabilization the cells remained viable and responded normally to cell signaling protein. We applied this technique to observe the dynamic motion of labeled actin and mitochondria using super-resolution fluorescence microscopy (dSTORM). Furthermore, we show the ability to image single proteins inside the living cell by tracking single molecules of kinesin using photostable probes.","abstract_html":"Fluorescence imaging of intracellular proteins is often achieved by using transfection-induced expression of fluorescent protein. This can potentially impose artifacts such as loss of function or over-expression of target proteins. Direct labeling of the intracellular protein is an alternative to transfection, but is largely limited by permeability of the fluorescent probes. Here, we have developed a high-throughput technique for labeling intracellular proteins of living cells. The technique makes use of Streptolysin O (SLO), a bacterial enzyme that permeabilizes cells to DNA, RNA, proteins. We show that SLO can be used to deliver a variety of fluorescent probes; ranging from organic dyes (&lt;1 kDa in size) to fluorescent immunoglobulin antibody (~150 kDa) for specific labeling of intracellular proteins. We demonstrate in numerous ways that after permeabilization the cells remained viable and responded normally to cell signaling protein. We applied this technique to observe the dynamic motion of labeled actin and mitochondria using super-resolution fluorescence microscopy (dSTORM). Furthermore, we show the ability to image single proteins inside the living cell by tracking single molecules of kinesin using photostable probes.","abstract_has_math":false,"creators":["Teng, Kai Wen"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Computnl Biology","degree_department":null,"school":null,"contributors":["Selvin, Paul R.","Zimmerman, Steven C.","Schroeder, Charles M.","Zhang, Kai"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-03-01T16:37:13Z","date_published":"2017-03-01T16:37:13Z","updated_at":"2026-07-22T22:26:37Z","subjects":["Fluorescence Microscopy","Live Cell Imaging","Protein Labeling","Reversible Membrane Permeabilization","Streptolysin O"],"languages":["en"],"rights":["Copyright 2016 Kai wen Teng"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/95526","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Selvin, Paul R.","Zimmerman, Steven C.","Schroeder, Charles M.","Zhang, Kai"]},{"key":"dc:creator","label":"Author","values":["Teng, Kai Wen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2017-03-01T16:37:13Z","2019-03-02T10:15:30Z","2016-07-20","2016-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Computnl Biology"]},{"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":["Fluorescence Microscopy","Live Cell Imaging","Protein Labeling","Reversible Membrane Permeabilization","Streptolysin O"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2016 Kai wen Teng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/95526"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Fluorescence imaging of intracellular proteins is often achieved by using transfection-induced expression of fluorescent protein. This can potentially impose artifacts such as loss of function or over-expression of target proteins. Direct labeling of the intracellular protein is an alternative to transfection, but is largely limited by permeability of the fluorescent probes. Here, we have developed a high-throughput technique for labeling intracellular proteins of living cells. The technique makes use of Streptolysin O (SLO), a bacterial enzyme that permeabilizes cells to DNA, RNA, proteins. We show that SLO can be used to deliver a variety of fluorescent probes; ranging from organic dyes (<1 kDa in size) to fluorescent immunoglobulin antibody (~150 kDa) for specific labeling of intracellular proteins. We demonstrate in numerous ways that after permeabilization the cells remained viable and responded normally to cell signaling protein. We applied this technique to observe the dynamic motion of labeled actin and mitochondria using super-resolution fluorescence microscopy (dSTORM). Furthermore, we show the ability to image single proteins inside the living cell by tracking single molecules of kinesin using photostable probes.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2018-12-01","The student, Kai Wen Teng, accepted the attached license on 2016-07-13 at 16:36.","The student, Kai Wen Teng, submitted this Dissertation for approval on 2016-07-13 at 16:46.","This Dissertation was approved for publication on 2016-07-20 at 17:08.","DSpace SAF Submission Ingestion Package generated from Vireo submission #9824 on 2017-02-28 at 14:35:24","Made available in DSpace on 2017-03-01T16:37:13Z (GMT). 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This can potentially impose artifacts such as loss of function or over-expression of target proteins. Direct labeling of the intracellular protein is an alternative to transfection, but is largely limited by permeability of the fluorescent probes. Here, we have developed a high-throughput technique for labeling intracellular proteins of living cells. The technique makes use of Streptolysin O (SLO), a bacterial enzyme that permeabilizes cells to DNA, RNA, proteins. We show that SLO can be used to deliver a variety of fluorescent probes; ranging from organic dyes (<1 kDa in size) to fluorescent immunoglobulin antibody (~150 kDa) for specific labeling of intracellular proteins. We demonstrate in numerous ways that after permeabilization the cells remained viable and responded normally to cell signaling protein. We applied this technique to observe the dynamic motion of labeled actin and mitochondria using super-resolution fluorescence microscopy (dSTORM). Furthermore, we show the ability to image single proteins inside the living cell by tracking single molecules of kinesin using photostable probes.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2018-12-01","The student, Kai Wen Teng, accepted the attached license on 2016-07-13 at 16:36.","The student, Kai Wen Teng, submitted this Dissertation for approval on 2016-07-13 at 16:46.","This Dissertation was approved for publication on 2016-07-20 at 17:08.","DSpace SAF Submission Ingestion Package generated from Vireo submission #9824 on 2017-02-28 at 14:35:24","Made available in DSpace on 2017-03-01T16:37:13Z (GMT). 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