Applications of pump-probe and two-photon techniques in time-resolved fluorescence miscroscopy and spectroscopy
Dong, Chen-Yuan
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https://hdl.handle.net/2142/30810
Description
Title
Applications of pump-probe and two-photon techniques in time-resolved fluorescence miscroscopy and spectroscopy
Author(s)
Dong, Chen-Yuan
Issue Date
1998-03
Doctoral Committee Chair(s)
Selvin, Paul R.
Gratton, E.
Committee Member(s)
Weissman, Michael B.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
pump-probe spectroscopy
two-photon fluorescence microscopy
harmonics
frequency spectrum analyzer
Language
en
Abstract
Pump-probe spectroscopy has long been useful in the elucidating ultrafast molecular phenomena. We developed a variation of time-resolved, pump-probe methodology using cross-correlation principles of the frequency-domain. An unique feature of our technique is that the low-frequency, cross correlation signal and its harmonics which contain lifetime information at high excitation frequencies can be simultaneously acquired and analyzed by a frequency spectrum analyzer. The result is that high frequency spectral information can be acquired without a fast optical detector and simultaneous acquisition of multiple harmonics reduce data acquisition time. A second key feature of this technique is that the cross-correlation signal comes primarily from the focal volume and hence microscopic imaging using this technique results in axial depth discrimination and improved spatial resolution similar in two-photon excitation microscopy. Using this technique, spectroscopic data measuring the lifetime and rotational correlation time of aqueous rhodamine B are presented. Furthermore, timeresolved, microscopic images deomonstrate this technique's capabilities in obtaining lifetimes and time-resolved polarization information of fluorescent molecules in mouse fibroblast (STO) cells. Two-photon fluorescence microscopy has proven to be an useful technique for imaging biological systems. Good spatial resolution, reduced photobleaching, and efficient rejection of excitation wavelength from emitted fluorescence are the mam characteristics of this technique. As part of this work, extension of two-photon microscopy to time-resolved polarization imaging of probe molecules inside STO cells are shown. Depending on the type of probe molecules and the nature of their interaction with cellular constituents, different degrees of rotational correlation times are obtained.
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