Fluorescence microscopy has been a fundamental imaging tool for life science research. Fluorescence basically involves only two molecular states: the ground molecular state and the first singlet excited state (the fluorescent state). Astonishingly, it greatly diversified the applicability of fluorescence microscopy in many different ways by incorporating additional molecular states and switching fluorescent molecules through these three or even more molecular states during the fluorescence process. This switching mechanism between additional molecular states, either long lifetime or short lifetime, and two original molecular states actually adds nonlinearity into the linear fluorescence process, which empowers fluorescence microscopy additional imaging capabilities. Herein, we developed four distinct new imaging techniques by taking advantage of this molecular switching mechanism: dark state dynamics sensing and imaging by fluorescence anomalous phase advance, genetically-encoded microviscosity sensor using protein-flexibility mediated photochromism, deep tissue imaging with super-nonlinear fluorescence microscopy, and light-driven fluorescent timer for simultaneous spatial-temporal mapping of protein dynamics in live cells.
The first technique, dark state dynamics sensing and imaging, effectively correlates the first triplet state of fluorescent organic dyes with the fluorescence process to produce fluorescence emission with unexpected phase advance compared with the excitation light, that reflects the real-time information of organic dyes' dark states. The last three techniques all harness the unique on-off switch capability of the optical highlighter fluorescent proteins: Dronpa, a photo-switchable fluorescent protein, is demonstrated to experience medium friction during the chromophore's cis-trans isomerization process while photo-switching from the bright state to the dark state; multiphoton fluorescence microscope could achieve higher order nonlinearity and thus deeper image depth in the scattering sample by the population transfer kinetics of the photoinduced molecular switches, such as photo-activatable fluorescent protein etc.; a photo-convertible fluorescent protein, mEos2, shows slow color conversion from green to red under extremely weak near-UV light, that could be used to time protein age. No matter fluorescent organic dyes or optical highlighter fluorescent proteins, the nonlinearity has been demonstrated to create new fluorescence imaging techniques by switching fluorescent molecules between additional molecular states and two original molecular states involved in the fluorescence process.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D85X2H8F |
| Date | January 2013 |
| Creators | Zhu, Xinxin |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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