Wang, Manyan
(2008)
SINGLE-CELL ELECTROPORATION USING ELECTROLYTE-FILLED CAPILLARIES WITH MICRO-SCALE TIPS.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Single-cell electroporation (SCEP) is a recently developing powerful technique for cell analysis and cell manipulation. In the first chapter of this thesis, a review about the theories and techniques is fulfilled, including a detailed description of the factors affecting SCEP, and a discussion about how to optimize SCEP for high efficiency and survivability. Based on the previous experimental results and numerical simulation, a hypothesis is proposed which leads us to find that small tips could be a solution to electroporate small cells with simultaneous maximization of electroporation efficiency and survivability when using electrolyte-filled capillaries (EFC) with pulled tips.In the second chapter, an integrated circuit for SCEP and controlling is demonstrated. EFCs with 2 &mu tips are constructed and used for SCEP of A549 cells with an extremely high spatial resolution. Distance between tip and cell is revealed to be vital in SCEP because of its direct control of the local electric field distribution and strength; to control distance precisely, a current measurement method inspired by tip-cell giga-seals is applied. High temporal resolution videos hint an abrupt intracellular fluorescence loss at the time scale of pulse duration followed by recovery in the small portion of cell membrane facing the tips. Viability of cells is highly related to the fluorescence loss, fluorescence exposure and dye types. Comsol simulation using the real shape capillaries helps to guide the electroporation throughout our experiments.However, this protocol evokes overcoming technical difficulties in terms of getting high survivability and decreasing variance, which are our two main aims. The advantage of small tips and the hypothesis are still to be examined. This is referred in the third chapter, as well as other following-up future work.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
28 September 2008 |
| Date Type: |
Completion |
| Defense Date: |
14 January 2008 |
| Approval Date: |
28 September 2008 |
| Submission Date: |
2 June 2008 |
| Access Restriction: |
5 year -- Restrict access to University of Pittsburgh for a period of 5 years. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
electrolyte-filled capillaries (EFC); single-cell; Electroporation; micro-tips |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-06022008-163731/, etd-06022008-163731 |
| Date Deposited: |
10 Nov 2011 19:46 |
| Last Modified: |
15 Nov 2016 13:44 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/7993 |
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