Photonic Imaging for Biology

Light microscopy is a central tool in biological research, allowing scientists to observe living cells and organisms with details invisible to the naked eye. Since its inception in the 17th century, it has evolved through key innovations in optics, staining, electronics and informatics. Major milestones include phase contrast, differential interference contrast, immunofluorescence, genetically encoded fluorescent proteins, confocal microscopy and super-resolution microscopy. The discovery of Green Fluorescent Protein (GFP) revolutionized molecular biology, while 21st-century advances, such as super-resolution microscopy and artificial intelligence, have pushed imaging capabilities even further. Modern microscopes now integrate digital imaging, advanced optics and computational analysis for enhanced visualization and interpretation. Photonic Imaging for Biology outlines major microscopy techniques that have driven biological discoveries, starting with fundamental principles and covering a range of methods, including brightfield, fluorescence, confocal, light-sheet, single particle tracking, photoperturbation, fluorescence correlation spectroscopy and super-resolution microscopy. The book concludes with a chapter on image analysis, highlighting recent progress in artificial intelligence. Each chapter focuses on specific techniques and their applications, strengths and limitations.

Preface xi Jean-Baptiste SIBARITA Chapter 1. Principles of Light Microscopy 1 Guillaume DUPUIS 1.1. Introduction 1 1.2. Principle of image formation 3 1.3. Optical sectioning techniques in fluorescence microscopy 23 1.4. Conclusion 32 1.5. References 32 Chapter 2. Contrast-based Label-Free Imaging and Phase Measurement 35 Pierre BON 2.1. Introduction, the biological object as an index object 35 2.2. Zernike phase contrast 38 2.3. Differential interference contrast 42 2.4. Other contrast methods with transparent objects 45 2.5. Measuring phase quantitatively: more than just contrast 46 2.6. Conclusions 49 2.7. References 49 Chapter 3. Fluorophores and Labeling Methods for Fluorescence Microscopy 51 Jip WULFFELÉ and Dominique BOURGEOIS 3.1. Introduction 51 3.2. Basics of fluorophore photophysics 52 3.3. Fluorescent proteins 53 3.4. Organic dyes 59 3.5. Conclusion 64 3.6. References 65 Chapter 4. Quantitative FRAP and FCS 69 Cyril FAVARD 4.1. Life is motion 69 4.2. FRAP 71 4.3. FCS 82 4.4. Conclusion 94 4.5. References 94 Chapter 5. Single-Particle Tracking for Nanoscale Dynamics of Biological Samples 97 Antony LEE and Laurent COGNET 5.1. Introduction 97 5.2. Nanoscale localization 98 5.3.Trajectory reconstruction 100 5.4. Nanoscale dynamics 102 5.5. References 110 Chapter 6. In Depth Microscopy 113 Tom DELAIRE and Rémi GALLAND 6.1. Introduction 113 6.2. Confocal microscopy 115 6.3. Multi-photon microscopy 120 6.4. Light-sheet fluorescence microscopy 125 6.5. Computational-based methods. 132 6.6. Futures challenges for 3D imaging of complex samples 134 6.7. References 139 Chapter 7. Structured Illumination Microscopy 145 Alexandra FRAGOLA 7.1. Introduction 145 7.2. The principle of structured illumination microscopy 146 7.3. Reconstructing the 2D SIM image 149 7.4. Experimental implementation of the 2D SIM 151 7.5. 3D SIM 153 7.6. Saturated SIM, or SSIM: a resolution gain above a factor of 2 154 7.7. Conclusion and perspective 155 7.8. References 157 Chapter 8. STED Imaging of Neuronal Morphology 159 Veera Venkata Gopala Krishna INAVALLI and Urs Valentin NÄGERL 8.1. Introduction 159 8.2. Principle 162 8.3. Instrumentation of STED 165 8.4. New developments 177 8.5. Summary and outlook 182 8.6. References 183 Chapter 9. Single-Molecule Localization Microscopy: From Imaging Cellular Structures to Quantitative Image Analysis 189 Marina S. DIETZ and Mike HEILEMANN 9.1. Introduction 189 9.2. Concepts for single-molecule localization microscopy 190 9.3. Multiplexing 193 9.4. Extracting quantitative information from SMLM data 196 9.5. Conclusion 198 9.6. Acknowledgments 198 9.7. References 198 Chapter 10. Image Processing and Image Analysis in Microscopy 205 Daniel SAGE and Anaïs BADOUAL 10.1. Introduction 205 10.2. Context 206 10.3. Biomage processing 213 10.4. Bioimage analysis 221 10.5. Discussion 230 10.6. References 232 List of Authors 239 Index 241