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Title:Intracellular labeling of live cells via transient membrane permeabilization for fluorescence and super resolution microscopy
Author(s):Teng, Kai Wen
Director of Research:Selvin, Paul R
Doctoral Committee Chair(s):Selvin, Paul R
Doctoral Committee Member(s):Zimmerman, Steven C; Schroeder, Charles M; Zhang, Kai
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Fluorescence Microscopy
Live Cell Imaging
Protein Labeling
Reversible Membrane Permeabilization
Streptolysin O
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.
Issue Date:2016-07-20
Type:Thesis
URI:http://hdl.handle.net/2142/95526
Rights Information:Copyright 2016 Kai wen Teng
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12


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