Developments of Two-Photon Fluorescent Probes for Amyloid-β Plaques and EGF-Conjugated Two-Photon Fluorescent Probes for Real-Time Monitoring of pH in Living System
Developments of Two-Photon Fluorescent Probes for Amyloid-β Plaques and EGF-Conjugated Two-Photon Fluorescent Probes for Real-Time Monitoring of pH in Living System
- 주제(키워드) two-photon microscopy , amyloid-β diagnosis , bio-conjugate , ratiometric pH probe
- 발행기관 아주대학교
- 지도교수 김환명
- 발행년도 2016
- 학위수여년월 2016. 8
- 학위명 박사
- 학과 및 전공 일반대학원 에너지시스템학과
- 실제URI http://www.dcollection.net/handler/ajou/000000023170
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Optical imaging with fluorescence microscopy is an important tool in biological and biomedical research. Recently, two-photon microscopy (TPM) has become a powerful tool for bio imaging. TPM utilizes low energy of two near-IR photons for excitation; it allows 1) higher spatial resolution images with minimum background emission, 2) reduced attenuation in biological specimens, 3) higher tissue penetration depth, and 4) prolonged observation time. However, the improvement in this field is limited by the lack of two-photon (TP) probes for specific cellular events. Therefore, there is a pressing need to explore various TP probes for specific applications. To meet such demands, we have developed TP probes that can detect amyloid-β plaques and pH in live cells and living mice. The outline of the present thesis, “Developments of two-photon fluorescent probes for amyloid-β plaques and two-photon bioconjugates for real-time monitoring of pH,” the abstract is as follows. Alzheimer’s disease (AD) is most common type of dementia that loss of brain function. AD is the accumulation of beta amyloid (Aβ) plaques and neurofibrillary tangles are thought to contribute to the degradation of the neurons in the brain. The Aβ plaques are a major pathogenesis of AD. Molecular imaging of Aβ plaques in vivo is a challenging task for early diagnosis and treatment of AD. Herein, we present new Aβ plaque-specific TP probes (SAD1, QAD1), which show a large significant TP action cross section, high selectivity for Aβ plaque specifically and readily crossed the blood brain barrier (BBB). Moreover, these probes can monitor direct 3D of the individual plaque at more than 400 µm deep inside of living mice brains with minimum background emission and photobleaching artifacts. The high photostability, low cytotoxicity, and excellent staining ability of these probes for amyloid plaques demonstrate that these probes are promising candidates for practical AD markers in biomedical research under the guidance of TPM. The acidic pH plays crucial roles in many cellular metabolisms, such as endocytic processes, signalling, apoptosis, and defence. Endocytosis is a type of active transport that moves particles, such as large molecules, parts of cells, and even whole cells, into a cell. It is an essential function in all cells and is required for nutrient uptake, receptor internalization, and synaptic transmission. The pH values change in the endocytic pathways, with acidification increasing from the endocytic vesicles and early endosomes to late endosomes and lysosomes. Herein, we developed a series of EGF-conjugates ratiometric TP probes for acidic pH (BH1-S series) that can perform real-time monitoring and quantitative analysis of acidic vesicles’ pH values during endocytic pathways. These probes are designed by a simple and flexible strategy, for acidic pH and conjugated with protein. Moreover, the ratiometric TPM imaging reveals that BH1-S3-EGF, an EGF conjugated pH probe for endocytosis, exhibits easy loading by EGFR, and thus allowing quantitative analysis of endocytic vesicles pH values during endocytosis. BH1-S3-EGF is able to monitor the change of acidic vesicles pH values during endocytosis along with their activity in live cells.
more목차
CHAPTER 1 1
1.1 GENERAL INTRODUCTION. 1
1.1.1 Two-Photon Absorption 1
1.1.2 Advantages of Two-Photon Microscopy 2
1.1.3 Design of Two-Photon Probes. 4
1.1.4 Two-Photon Probes for Bioimaging Applications 6
1.2 REFERENCES. 8
CHAPTER 2 9
2.1 A Two-Photon Fluorescent Probe for Amyloid-β Plaques in Living Mice 10
2.1.1 INTRODUCTION 10
2.1.2 RESULTS AND DISCUSSION. 11
2.1.2.1 Design and Preparation of SAD1 11
2.1.2.2 Photophysical Properties of SAD1 12
2.1.2.3 TPM images with SAD1 18
2.1.3 CONCLUSION. 23
2.1.4 EXPERIMENTAL SECTION. 24
2.1.5 REFERENCES 30
2.2 A Quadrupolar Two-Photon Fluorescent Probe for in vivo Imaging of Amyloid-β Plaques 33
2.2.1 INTRODUCTION 33
2.2.2 RESULTS AND DISCUSSION. 34
2.2.2.1 Design and Preparation of QAD1. 34
2.2.2.2 Photophysical Properties of QAD1. 35
2.2.2.3 TPM images with QAD1 44
2.2.3 CONCLUSION. 50
2.2.4 EXPERIMENTAL SECTION. 51
2.2.5 REFERENCES 63
CHAPTER 3 65
3.1 EGF-Conjugated Ratiometric Two-Photon Probes for Real-Time Monitoring of pH Values during Endocytic Pathway 66
3.1.1 INTRODUCTION 66
3.1.2 RESULTS AND DISCUSSION. 68
3.1.2.1 Design and Preparation of QAD1. 68
3.1.2.2 Photophysical Properties of QAD1. 70
3.1.2.3 EGF-conjugation of BH1-S series 74
2.1.2.4 TPM images with BH1-S3-EGF. 77
3.1.3 CONCLUSION. 80
3.1.4 EXPERIMENTAL SECTION. 81
3.1.5 REFERENCES 90
APPENDIX. 93
CURRICULUM VITAE. 105
