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Methodology development and biologic...

University of California, Los Angeles.

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Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy./
作者:	Korlann, You.
面頁冊數:	139 p.
附註:	Adviser: Shimon Weiss.
Contained By:	Dissertation Abstracts International70-03B.
標題:	Biophysics, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3350548
ISBN:	9781109057638

Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy.
Korlann, You.

Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy. - 139 p.

Adviser: Shimon Weiss.

Thesis (Ph.D.)--University of California, Los Angeles, 2008.

In this dissertation, methodology developments in fluorescence spectroscopy and microscopy, and their applications to biological questions will be presented. First, three methodology advancements of the alternating laser excitation (ALEX) technique developed in our laboratory will be detailed. ALEX advances conventional single-pair fluorescence resonance energy transfer (FRET) measurements between a donor and acceptor dye pair by adding a second laser that directly excites the acceptor to enable simultaneous analysis of biomolecular structures and interactions at single molecule level. In order to improve the distance information provided by ALEX FRET measurements, we first introduced a method to accurately measure FRET efficiencies between the donor and acceptor molecules, independent of instrumental factors. Donor-acceptor distances on DNA fragments determined by this method were shown to fit theoretical values better than distances obtained at the ensemble level. Then, ALEX was extended further to allow three spectrally distinct fluorescent probes to be monitored simultaneously (3c-ALEX). This technique sorts, identifies and selects fluorescent species with different probe-stoichiometries, and accurately generates up to three inter-probes distances simultaneously. Downstream translocation of E. coli RNA polymerase on DNA was monitored from two perspectives within a triply labeled transcription complex. Finally, ALEX was combined with total-internal-reflection (TIR) fluorescence microscopy to track single fluorescent molecules beyond the time limited by diffusion in the solution-based measurements. Abortive initiation and promoter escape within single immobilized transcription complex was detected with this methodology, and results confirm and extend those obtained from the single diffusing molecules.

ISBN: 9781109057638Subjects--Topical Terms:

1019105
Biophysics, General.

Methodology development and biological applications of single molecule fluorescence spectroscopy and microscopy.
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520 $a In this dissertation, methodology developments in fluorescence spectroscopy and microscopy, and their applications to biological questions will be presented. First, three methodology advancements of the alternating laser excitation (ALEX) technique developed in our laboratory will be detailed. ALEX advances conventional single-pair fluorescence resonance energy transfer (FRET) measurements between a donor and acceptor dye pair by adding a second laser that directly excites the acceptor to enable simultaneous analysis of biomolecular structures and interactions at single molecule level. In order to improve the distance information provided by ALEX FRET measurements, we first introduced a method to accurately measure FRET efficiencies between the donor and acceptor molecules, independent of instrumental factors. Donor-acceptor distances on DNA fragments determined by this method were shown to fit theoretical values better than distances obtained at the ensemble level. Then, ALEX was extended further to allow three spectrally distinct fluorescent probes to be monitored simultaneously (3c-ALEX). This technique sorts, identifies and selects fluorescent species with different probe-stoichiometries, and accurately generates up to three inter-probes distances simultaneously. Downstream translocation of E. coli RNA polymerase on DNA was monitored from two perspectives within a triply labeled transcription complex. Finally, ALEX was combined with total-internal-reflection (TIR) fluorescence microscopy to track single fluorescent molecules beyond the time limited by diffusion in the solution-based measurements. Abortive initiation and promoter escape within single immobilized transcription complex was detected with this methodology, and results confirm and extend those obtained from the single diffusing molecules.
520 $a In addition, to advancing single-molecule techniques, we also developed improved methodology for fluorescence correlation spectroscopy (FCS), where both single and several fluorescent molecules can be monitored. FCS allows continuous observation for longer periods of time without requiring selection of specific molecules for observation. I present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently introduced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations.
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