Intracellular labeling of live cells via transient membrane permeabilization for fluorescence and super resolution microscopy

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Computnl Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2017

Author dc:creator

• Teng, Kai Wen

Contributors dc:contributor

• Selvin, Paul R.
• Zimmerman, Steven C.
• Schroeder, Charles M.
• Zhang, Kai

• Fluorescence Microscopy
• Live Cell Imaging
• Protein Labeling
• Reversible Membrane Permeabilization
• Streptolysin O

Statement dc:rights

• Copyright 2016 Kai wen Teng

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/95526

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/95526