細胞代謝在調節正常細胞功能和維持能量平衡中至關重要。氧消耗率（OCR）對監測細胞代謝至關重要。傳統的細胞分析方法以大批量細胞測量為特徵，往往未能捕捉個體細胞之間的異質性。本論文探討創新的方法來解決這一差距。該研究的主要目的是開發和驗證結合了寬場頻域螢光生命週期成像顯微術（FD-FLIM）的微流體設備。這些設備旨在實現高效率單細胞和類球體OCR的表徵，從而以高精度的方式展現細胞功能和異質性。研究的第一部分介紹了一種用於單細胞OCR分析的微流體設備。該設備將細胞隔離在含有氧敏感磷光染料的微孔中，能夠精確測量MCF-7乳腺癌細胞的OCR。研究的第二部分詳細介紹了另一種用於三維（3D）類球體形成、培養和氧消耗分析的微流體平台。該設備用於測試MG-63骨肉瘤細胞，允許在非侵入性方法造成結構損害的情況下，對藥物治療的氧消耗率進行準確的量測。這兩種設備在各自的應用中都展示了卓越的功能：單細胞分析揭示了MCF-7細胞之間的顯著異質性，而類球體分析提供了MG-63細胞的準確氧消耗測量，了解了細胞對藥物的反應。這些發現顯示了所開發方法在細胞代謝研究中的有效性，展現了將微流體設備與FD-FLIM整合對細胞代謝研究的一項重大進展，提供了具有高靈敏度的單類球體和單細胞分辨能力。

Cell metabolism is essential in regulating normal cell functions and maintaining energy homeostasis. The oxygen consumption rate (OCR) is critical in monitoring cell metabolism. Traditional cell analysis methods, characterized by bulk cell measurement, often fail to capture the heterogeneity among individual cells. This thesis explores innovative approaches to address this gap. The primary objective of this research is to develop and validate microfluidic devices integrated with widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM). These devices aim to enable efficient, single-cell, and spheroid OCR characterization, thereby revealing cellular functions and heterogeneity with high precision. The first part of the study introduces a microfluidic device designed for single-cell OCR analysis. This device isolates cells in microwells containing an oxygen-sensitive phosphorescent dye, enabling the precise measurement of OCR in MCF-7 breast cancer cells. The second part of the study details the development of another microfluidic platform for three-dimensional (3D) spheroid formation, culture, and oxygen consumption analysis. This device, tested with MG-63 osteosarcoma cells, allows for accurate OCR characterization in response to drug treatments without the structural damage common in invasive methods. Both devices demonstrated remarkable functionality in their respective applications. The single-cell analysis revealed significant heterogeneity among MCF-7 cells, while the spheroid analysis provided accurate oxygen consumption measurements of MG-63 cells, emphasizing the cellular response to pharmacological interventions. These findings underline the efficacy of the developed approaches in cellular metabolism studies. Integrating microfluidic devices with FD-FLIM represents a significant advancement in cellular metabolism research, offering single spheroid and single-cell resolution with high sensitivity.

Contents
Chapter 1 Introduction..........................................1
1.1 Background..................................................1
1.1.1 Importance of Oxygen in Biological Systems................1
1.1.2 Challenges of In Vivo Measurement.........................1
1.1.3 Monitoring Oxygen Consumption: The In Vitro Advantage.....2
1.2 Motivation..................................................3
1.3 Specific aims...............................................5
1.4 Thesis Outline..............................................6
Chapter 2 Literature Review.....................................9
2.1 Cell Metabolism.............................................9
2.1.1 The Role of Oxygen in Human Bodies........................9
2.2 In vitro models............................................12
2.3 Comparison of 2D and 3D Cell Culture Models................16
2.3.1 Impact on Proliferation, Differentiation, and Gene Expression ..................................................................17
2.3.2 Apoptosis, Cell Motion, and Migration....................18
2.3.3 Extracellular Matrix, Mechanical Responses, and Cell Behavior ..................................................................19
2.4 Applications and Advancements in 3D Tumor Models...........21
2.5 Oxygen Sensing Methods.....................................22
2.5.1 Fluorescence-Based Methods...............................24
2.5.2 Quenching of Excited Metal Complexes by Oxygen...........26
2.5.3 Intensity and Lifetime-Based Methods.....................27
Chapter 3 Frequency Domain – Fluorescence Lifetime Imaging Microscopy (FD-FLIM) for oxygen sensing...........................................................29
3.1 Introduction...............................................29
3.2 Working Principle..........................................30
3.3 FLIM Camera System.........................................32
3.4 Experimental Procedures....................................33
3.5 Oxygen Tension Calculation.................................34
3.6 FD-FLIM Image Analysis.....................................37
3.7 Results and Discussions....................................39
3.8 Conclusion.................................................43
Chapter 4 Efficient single-cell Oxygen Consumption Rate Characterization Based on Frequency Domain Fluorescence Lifetime Imaging Microscopy Measurement and Microfluidic Platform..........................................................44
4.1 Introduction...............................................44
4.2 Materials and Methods......................................53
4.2.1 Microfluidic Device Design...............................53
4.2.2 Device Fabrication.......................................57
4.2.3 Cell Culture.............................................58
4.2.4 Experimental Procedure...................................58
4.2.5 Cell Culture and Analysis................................61
4.2.6 OCR Calculation..........................................63
4.2.7 Statistical Analysis.....................................63
4.3 Results and Discussion.....................................63
4.3.1 Single-Cell Trapping and Isolation.......................63
4.3.2 Single-Cell OCR Measurement..............................66
4.4 Conclusion.................................................74
Chapter 5 Characterization of Single Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy........................................................75
5.1 Introduction...............................................75
5.2 Materials and Methods......................................80
5.2.1 Microfluidic Device Design and Fabrication...............80
5.2.2 Cell Culture.............................................82
5.2.3 Tumor Spheroid Formation.................................82
5.2.4 Cell viability...........................................85
5.2.5 Oxygen Tension Measurement...............................86
5.2.6 Image Analysis...........................................87
5.2.7 Drug Treatment...........................................88
5.3 Results and Discussion.....................................89
5.3.1 Formation and Culture of Single Spheroids................89
5.3.2 Spheroid Size Optimization...............................92
5.3.3 Cell Viability and Spheroid Growth Analysis..............94
5.3.4 Oxygen Tension Measurement in the Microfluidic Device....98
5.3.5 Oxygen Consumption of the Single Spheroids...............101
5.4 Conclusions................................................105
Chapter 6 Conclusion and Future Works..........................106
Bibliography......................................................109
Appendix A........................................................118

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