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Development of Two-Photon Probes for β-galactosidase Activity and Vesicular Transport in Live Sample

초록/요약

To fully understand the biological and medical phenomena, observation of live cells and tissues is very important. One-photon microscopy (OPM), which is currently the most widely used in fluorescence related study, has a low permeability to live samples as it uses short wavelength with high energy excitation sources. It might cause damage to biological specimens, and has low penetration depth with auto-fluorescence of the samples to limit their use to live cells and tissues. In recent years, two-photon microscopy (TPM) has been developed using light in the near-infrared region of low energy as an excitation source, contributing to the development of biological and medical research. TPM uses two low-energy photons as a light source to obtain a high-resolution image deep in the live specimens, and allows a long-time imaging with low damage to live cells and tissues. β-galactosidase (β-gal) is an essential enzyme widely found in animal cells, bacteria, yeast and higher plants, and hydrolyzes galactoside bonds of various physiologically active substances to produce galactose. The activity of β-gal is known to be particularly high in senescent cells and tissues, and senescence-associated β-gal (SA-β-gal) has been used as a representative senescence biomarker. Therefore, development of a fluorescent probe capable of selectively and sensitively detecting β-gal is highly promising. Herein, we developed a ratiometric fluorescent β-gal probe (SG1), which shows emission color change (blue to yellow) after β-gal enzymatic hydrolysis. This probe enables real-time, quantitative detection of β-gal activity, and it shows fine distinction of SA-β-gal activity in live cells and tissues. This probe might be helpful to the development of various research field including senescence-related disease. Material transport in cells occurs in a variety of ways, among which vesicular transport is responsible for the transport of proteins and lipids and plays an important role in many cellular metabolisms. Proteins or lipids produced in the endoplasmic reticulum (ER) are transported via Golgi apparatus to required organelles through various transport channels and mechanisms. The vesicular transport malfunction affects diseases such as arteriosclerosis, Alzheimer's disease and various genetic diseases. However, the mechanism of such vesicular transport is not yet clear. To develop probes capable of monitoring vesicular transport in steady and diseased state by imaging in real time, we designed a small molecule amidine-derived two-photon fluorescent probe (ELP1) suitable for tracking vesicular transport in live cells. Using this probe, we successfully visualize the vesicular transport from the ER to lysosomal compartments with TPM, and this could be a useful tool for biomedical research such as ER-related biology and cellular pathology associated with vesicular transport.

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목차

CHAPTER 1 1
1.1 GENERAL INTRODUCTION 1
1.1.1 Two-Photon Absorption and Two-Photon Microscopy 1
1.1.2 Design of Two-Photon Fluorescent Probes 4
1.1.3 Variety of Two-Photon Sensing Mechanism 5
1.1.4 Two-Photon Probes for Biomedical Applications 6
1.2 REFERENCES 8
CHAPTER 2 10
2.1 A Ratiometric Two-Photon Probe for β-Galactosidase Activity and its Application to Senescent Cells 11
2.1.1 INTRODUCTION 11
2.1.2 RESULTS AND DISCUSSION 12
2.1.2.1 Design and Synthesis of SG1 12
2.1.2.2 Photophysical Properties of SG1 14
2.1.2.3 Enzymatic Reaction and Selectivity of SG1 17
2.1.2.4 TPM images of SG1 in live cells and tissues 22
2.1.3 CONCLUSION 33
2.1.4 EXPERIMENTAL SECTION 34
2.1.5 REFERENCES 44
CHAPTER 3 47
3.1 Small Molecule Two-Photon Probe for Visualization of Vesicular Transport from the Endoplasmic Reticulum to Lysosome 48
3.1.1 INTRODUCTION 48
3.1.2 RESULTS AND DISCUSSION 50
3.1.2.1 Design and Synthesis of ELP1 50
3.1.2.2 Photophysical Properties of ELP1 52
3.1.2.3 TPM images of ELP1 in live cells 54
3.1.3 CONCLUSION 62
3.1.4 EXPERIMENTAL SECTION 63
3.1.5 REFERENCES 69
APPENDIX 72
CURRICULUM VITAE 88
ABSTRACT IN KOREAN 93

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