Table of Contents

Preface vii

1 Fluorescent Protein Labeling Techniques Zhihong Zhang Qingming Luo 1

1.1 Introduction 1

1.2 Fluorescent proteins and their mutants 2

1.2.1 Colorful fluorescent proteins 3

1.2.2 Fluorescent proteins with LSSs 6

1.2.3 Photon-activatable and photon-switchable fluorescent proteins 7

1.2.4 Light-sensitive fluorescent proteins 9

1.2.5 Timer fluorescent protein 10

1.3 Reporter fluorescent protein probes 11

1.3.1 Tracking proteins in live cells 11

1.3.2 Monitoring of gene expression in live cells 14

1.3.3 Biological applications of photon-switchable proteins and photon-activatable proteins 15

1.4 Functional fluorescent protein probes 18

1.4.1 Redox probes 18

1.4.2 ATP fluorescent protein probes 21

1.4.3 pH probes 22

1.4.4 Voltage-sensitive probes 24

1.4.5 Calcium probes 26

1.4.6 Mercury ion probes 30

1.4.7 Copper ion probes 30

1.4.8 Zinc ion probes 31

1.5 Fluorescence resonance energy transfer (FRET) probes 32

1.5.1 Introduction of FRET 32

1.5.2 FRET imaging in cell biology research 34

1.5.3 Intramolecular FRET probes 36

1.5.4 Intermolecular FRET probes 40

1.6 BiFC technology based on fluorescent proteins 43

1.6.1 Establishment of the BiFC detection method 43

1.6.2 Characteristics of BiFC technology 44

1.6.3 Applications of BiFC technology 46

1.6.4 Quantitative detection of protein interaction based on fluorescence signal of BiFC 48

1.6.5 Limiting factors of bimolecular fluorescent complementation 48

1.6.6 Outlook for BiFC 49

1.7 Intravital applications of fluorescent proteins in tumor imaging 49

1.7.1 In vivo tumor optical imaging based on endogenously expressed fluorescent protein 50

1.7.2 Optical imaging of tumor in vivo with targeting FP probes 59

1.7.3 Prospects 62

1.8 Applications of fluorescent protein transgenic mice in intravital immune optical imaging 63

1.8.1 Fluorescent protein transgenic animal models 63

1.8.2 Applications of fluorescent protein-labeled pathogens in infection and immune imaging 68

Bibliography 74

2 Two-photon Molecular Probe Yu Li Lingyu Zeng Zhihong Liu Jingui Qin 93

2.1 Introduction of two-photon absorption 93

2.1.1 The basic concept of 2PA 93

2.1.2 Measurements of 2PA effect 96

2.1.3 Introduction to application of 2PA effect 99

2.2 Molecular design and structure-property relationships of organic TPA materials 103

2.2.1 One-dimensional asymmetric D-π-A molecules 104

2.2.2 One-dimensional symmetric molecules 107

2.2.3 Porphyrins and expanded porphyrinoids 112

2.2.4 Multidimensional branched 2PA materials 116

2.3 The development of two-photon fluorescent probes 119

2.3.1 Brief introduction to response principle of fluorescent probes 120

2.3.2 Traditional fluorescent probes for two-photon imaging 122

2.3.3 Typical fluorophores for TP probes 122

2.3.4 Research development of TP probes 125

2.3.5 Research prospection of TP probes 181

Acknowledgment 186

Bibliography 186

3 Super-resolution Localization Microscopy Zhenli Huang Yina Wang Fan Long Zhe Hu Zeyu Zhao 194

3.1 Introduction and background 194

3.1.2 Resolution limit of optical microscope 196

3.1.3 Improving the resolution of optical microscope 197

3.1.4 A historical overview of super-resolution localization microscopy 197

3.1.5 Breaking the resolution limit by single-molecule localization 199

3.2 Fluorescence probes for super-resolution localization microscopy 200

3.2.1 Ensemble and single-molecule fluorescence 200

3.2.2 Fluorescence probes and specific labeling 202

3.2.3 Fluorescence ON/OFF control 204

3.2.4 Choosing the right fluorescence probes 205

3.3 Methods and instrumentation in super-resolution localization microscopy 208

3.3.1 Super-resolution localization microscopy methods: PALM versus STORM 208

3.3.2 Super-resolution localization microscopy methods: Others 210

3.3.3 Instrumentation in super-resolution localization microscopy: Basic structure 210

3.3.4 Instrumentation in super-resolution localization microscopy: Key components 211

3.3.5 Instrumentation in super-resolution localization microscopy: A typical setup 214

3.3.6 Advances in super-resolution localization microscopy: Multicolor and 3D imaging 216

3.3.7 Commercial super-resolution localization microscopes 217

3.4 Data analysis in super-resolution localization microscopy 218

3.4.1 Theoretical localization precision 218

3.4.2 Practical aspects for determining spatial resolution 219

3.4.3 Single-molecule localization for sparse emitters 220

3.4.4 Single-molecule localization for high-density emitters 223

3.4.5 Key steps in image analysis and reconstruction 225

3.4.6 Data analysis software 226

3.5 Example applications in super-resolution localization microscopy 227

3.5.1 Imaging in 2D 227

3.5.2 Imaging in 3D 228

3.6 Conclusions and future prospects 229

Bibliography 230

4 Photoacoustic Molecular (Functional) Imaging Da Xing Sihua Yang 235

4.1 Introduction 235

4.2 PAI principle, algorithm, and system 238

4.2.1 PAI principle 238

4.2.2 Excitation of photoacoustic signal 239

4.2.3 Photoacoustic scanning method and its imaging algorithm 247

4.2.4 PAI system 254

4.2.5 Special problems involved 270

4.3 Domestic and foreign statuses 275

4.3.1 Foreign research status 275

4.3.2 Domestic research status 286

4.4 Application development trend 298

4.4.1 Application research of photoacoustic microcirculation imaging and early tumor detection and treatment monitoring 299

4.4.2 Research on application of living body photoacoustic blood function parameters (blood oxygen and carbon oxygen saturation) detection 306

4.4.3 Application research on photoacoustic identification and imaging of vulnerable plaque components in blood vessels 309

4.4.4 Application research on thermoacoustic imaging in testing of low-dentistry foreign bodies 313

4.4.5 Application research on thermoacoustic imaging in testing of breast cancer 315

Bibliography 318

5 Optical Molecular Imaging for Small Animals in vivo Yong Deng Xiaoquan Yang Qingming Luo 324

5.1 Models of light propagation in tissue 324

5.1.1 Introduction 324

5.1.2 Light transport equation 325

5.1.3 Diffusion approximation method 329

5.1.4 Monte Carlo method 333

5.2 Diffuse optical tomography 343

5.2.1 Introduction 343

5.2.2 DOT mode 344

5.2.3 Image reconstruction methods in DOT 348

5.2.4 Applications in biomedical research 352

5.3 In vivo optical molecular imaging of small animals 354

5.3.1 Introduction 354

5.3.2 Planar fluorescence molecular imaging 355

5.3.3 Fluorescence molecular tomography 361

5.3.4 Bioluminescence tomography 370

5.4 Multimodality molecular imaging of small animals in vivo 375

5.4.1 Introduction 375

5.4.2 Multimodality molecular imaging systems 376

5.4.3 Image reconstruction and multimodal image fusion 382

5.4.4 Applications in biomedical research 387

Bibliography 393

Index 402