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Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single- Molecule Imaging

Citation

Lee, Dongkwan (2025) Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single- Molecule Imaging. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8qqq-z286. https://resolver.caltech.edu/CaltechTHESIS:02282025-172449360

Abstract

Advances in optical microscopy have revolutionized cell biology, transforming our understanding of cellular processes from static structural observations to dynamic temporal and spatial insights at the single-molecule level. While fluorescence imaging remains the gold standard due to its high sensitivity, specificity, and versatile toolbox, it faces significant limitations, particularly in imaging small molecules that are not inherently fluorescent. Attaching fluorescent tags to these molecules often disrupts their physicochemical properties, highlighting the need for minimally invasive and intrinsic-contrast-based approaches.

Vibrational spectro-microscopy, which probes the intrinsic vibrational frequencies of chemical bonds, offers a promising solution. Stimulated Raman scattering (SRS) microscopy, a well-established vibrational imaging technique, enhances vibrational excitation by up 10⁸-fold through stimulated emission amplification, enabling rapid, label-free imaging of biological samples with high specificity.

In the first half of this thesis, we advance SRS microscopy to tackle specific biological challenges and explore new methodological possibilities. To visualize glycogen metabolism, we combined a stable isotope labeling strategy with SRS imaging, achieving high-specificity imaging of glycogen in live cells. This approach was further applied to metabolic phenotyping of patient-derived melanoma cell lines. Additionally, we investigated strategies to photoswitch electronic pre-resonance (epr) SRS probes, which are typically photostable. By inducing electronic transitions that modulate electronic-vibrational coupling, we developed the first genetically encodable photoswitchable epr-SRS probe using a near-infrared fluorescent protein, unlocking new possibilities in Raman imaging.

In the second half of this thesis, we address the limitations of SRS microscopy by developing a novel bond-selective nonlinear optical microscopy technique called bond-selective fluorescence-detected infrared-excited (BonFIRE). BonFIRE introduces a vibration-state-mediated two-photon process as a new vibrational contrast mechanism, overcoming key limitations in sensitivity and speed associated with SRS. By combining the high sensitivity and specificity of fluorescence with the rich chemical information provided by IR absorption-based vibrational contrast, BonFIRE offers a powerful platform for multidimensional insights into biological systems. We envision BonFIRE as a tool to tackle unique challenges that current technologies cannot address, representing a significant step forward in understanding the complex processes that define life.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Nonlinear optics; microscopy; spectroscopy; single molecule; stimulated Raman; chemical imaging
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Wei, Lu
Thesis Committee:
  • Shapiro, Mikhail G. (chair)
  • Tirrell, David A.
  • Wang, Lihong
  • Wei, Lu
Defense Date:31 January 2025
Funders:
Funding AgencyGrant Number
NIH Director’s New Innovator AwardGM140919
California Institute of TechnologyStartup funds
Record Number:CaltechTHESIS:02282025-172449360
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:02282025-172449360
DOI:10.7907/8qqq-z286
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acs.analchem.0c02348DOIArticle adapted for Chapter 2
https://doi.org/10.1063/5.0043791DOIArticle adapted for Chapter 3
https://doi.org/10.1038/s41566-023-01243-8DOIArticle adapted for Chapter 4
https://doi.org/10.1364/OPTICA.545195DOIArticle adapted for Chapter 5
https://doi.org/10.1021/acs.jpclett.4c00597DOIPublication mentioned in Chapter 1
https://doi.org/10.1002/ange.202413647DOIPublication mentioned in Chapter 1
https://doi.org/10.1021/cbmi.3c00004DOIPublication mentioned in Chapter 1
ORCID:
AuthorORCID
Lee, Dongkwan0000-0001-6091-1349
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:17035
Collection:CaltechTHESIS
Deposited By: Dongkwan Lee
Deposited On:05 Mar 2025 17:33
Last Modified:12 Mar 2025 20:21

Thesis Files

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[img] Video (AVI) (Movie S1) - Supplemental Material
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[img] Video (AVI) (Movie S2) - Supplemental Material
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[img] Video (AVI) (Movie S3) - Supplemental Material
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[img] Video (AVI) (Movie S4) - Supplemental Material
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