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Bond-Selective Nonlinear Optical Mic...

Lee, Dongkwan,

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

Record Type:	Electronic resources : Monograph/item
Title/Author:	Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single-Molecule Imaging // Dongkwan Lee.
Author:	Lee, Dongkwan,
Description:	1 electronic resource (181 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 86-12, Section: B.
Contained By:	Dissertations Abstracts International86-12B.
Subject:	Cancer. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=32006578
ISBN:	9798283492926

Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single-Molecule Imaging /
Lee, Dongkwan,

Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single-Molecule Imaging /Dongkwan Lee. - 1 electronic resource (181 pages)

Source: Dissertations Abstracts International, Volume: 86-12, Section: B.

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, labelfree 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 nearinfrared 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-statemediated 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 absorptionbased 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.

English

ISBN: 9798283492926Subjects--Topical Terms:

634186
Cancer.

Bond-Selective Nonlinear Optical Microscopy: From Live Cells to Single-Molecule Imaging /
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520 $a 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, labelfree 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 nearinfrared 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-statemediated 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 absorptionbased 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.
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