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Development of Genetically Targeted Voltage Sensitive Dyes
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Development of Genetically Targeted Voltage Sensitive Dyes. |
| 개인저자 | Liu, Pei. |
| 단체저자명 | University of California, Berkeley. Chemistry. |
| 발행사항 | [S.l.]: University of California, Berkeley., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 268 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-06B. Dissertation Abstract International |
| ISBN | 9781392801420 |
| 학위논문주기 | Thesis (Ph.D.)--University of California, Berkeley, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-06, Section: B.
Advisor: Miller, Evan W. |
| 이용제한사항 | This item must not be sold to any third party vendors. |
| 요약 | Comprehensively mapping and recording the electrical inputs and outputs of multiple neurons simultaneously with cellular spatial resolution and millisecond time resolution remains an outstanding challenge in the field of neurobiology. Development of fluorescence indicators for direct visualization of membrane potential changes offers a powerful method for probing voltage dynamics in neurons with unprecedented spatial and temporal resolution. Despite the promise of voltage imaging, key challenges of speed, sensitivity, brightness, and localization remain. The Miller lab has developed a new class of small molecules dyes, VoltageFluors (VF), that rely on photoinduced electron transfer (PeT) and display voltage-sensitive fluorescence due to modulation of the rate of PeT within the VF scaffold by the transmembrane electric field. This approach allows for fast, sensitive and non-invasive recording of neuronal activity in cultured mammalian neurons and in ex vivo tissue slices in single trials. One major limitation of small-molecule dye imaging is the inability to target the dye to specific cells of interest, which significantly erodes signal to noise and cellular resolution. To solve this problem, we have worked to combine VF dyes with a genetically encoded component to enable high-contrast imaging in defined neurons. We first attempted a fluorogenic approach, in which the parent VF dye is chemically modified to be minimally fluorescent and non-voltage-sensitive and must be enzymatically activated prior to imaging. We designed a couple of dyes which can only be activated by a cell-surface pig liver esterase (PLE) and observed enhanced contrast in both HEK cells and cultured neurons with good sensitivity. We have also shown that our genetically targeted voltage dyes outperform purely genetically encoded voltage indicators. In addition, we are exploring other enzyme/substrate pair for fluorogenic activation purpose. The second approach is covalent labeling of voltage sensitive dye to cells of interest using a cell-surface HaloTag enzyme. We have successfully demonstrated selective membrane staining in mouse brain slices and live animal using this strategy. Current efforts focus on applying this method for targeted voltage imaging in live animals to probe specific neuroscience questions. |
| 일반주제명 | Chemistry. |
| 언어 | 영어 |
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