Vibrational imaging approaches including Raman microscopy and IR-absorption micro-spectroscopy can provide rich chemical information about biological samples. This dissertation contributes to improve the capabilities of vibrational microscopy in three aspects each with corresponding biological applications. First, along the line of combining electronic resonant effect with stimulated Raman scattering (SRS), we studied the spectroscopic characteristics for on-resonant SRS case and demonstrated the utility of electronic pre-resonant SRS on super-multiplexed imaging for live cells and tissue sections. Second, we provided a new light-matter interaction as a hybrid technique of Raman and fluorescence, called stimulated Raman excited fluorescence (SREF), bringing the long-sought-after goal of detecting single-molecule Raman scattering without plasmonic enhancement into view. Coupling SREF with vibrational sensing, local electric field and hydrogen-bonding environment can thus be visualized in situ. Third, we brought small vibrational probes into mid-infrared imaging for the goal of rich-information-content, high-throughput metabolic imaging.
Chapter 1 introduces some basis of Raman scattering, and provides an overview of state-of-art SRS microscopy.
Chapter 2 explores on the rigorous electronic resonant region with SRS (er-SRS) through suppression of electronic background and subsequent retrieval of vibrational peaks. In agreement with theoretical prediction, changing of vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian is observed when approaching electronic resonance. As large as 10-23 cm2 of resonance Raman cross section is estimated in er-SRS.
In Chapter 3, a new light-matter interaction called stimulated Raman excited fluorescence (SREF) is studied. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement.
Chapter 4 details the development of SREF into a novel water-sensing tool, by coupling with vibrational solvatochromism of environment-sensitive Raman mode. This new technique allows direct visualization on spatially-resolved distribution of water states inside single mammalian cells. Interesting intracellular heterogeneity of water states between nucleus and cytoplasm has been revealed.
Chapter 5 demonstrates the utility of epr-SRS in super-multiplexed imaging with either commercial fluorophores in lives cells or our MARS probes on tissue sections. Multiplex protein-based tissue imaging is completed with newly-designed functional MARS dye with up to 12 channels simultaneously.
Chapter 6 focus on metabolic imaging by mid-infrared (MIR) microscopy with vibrational probes. Raman scattering microscopy has made a major advance in metabolic imaging utilizing vibrational probes, yet is limited to relatively low throughput. As an alternative solution, MIR microscopy provides significantly higher cross section and exhibits as a rich-information-content, high-throughput technique with recent rapid technical advances. We introduced three types of small vibrational probes as azide, 13C and carbon-deuterium for studying dynamic metabolic activities of protein, lipids and carbohydrates in cells, small organisms and mice for the first time. Two MIR microscopy platforms as Fourier transform infrared (FTIR) absorption microscopy and discrete frequency mid-infrared (DFIR) microscopy were utilized to validate the generality of our vibrational probes and applicability for single-cell metabolic profiling and metabolism study on large-scale tissues.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-85zn-4p12 |
| Date | January 2020 |
| Creators | Shi, Lixue |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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