Two-photon excited fluorescence (2PEF)

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Table of contents

1 Collagen 
1.1 Structure and synthesis
1.1.1 Collagen types
1.1.2 Collagen type I structure and synthesis
1.1.3 Hierarchical organization of collagen assemblies
1.2 Tendon biomechanics
1.3 Collagen visualization techniques and challenges
1.3.1 Conventional visualization techniques
1.3.1.1 Electron microscopy
1.3.1.2 Atomic force microscopy
1.3.1.3 Polarized light microscopy
1.3.1.4 Optical coherence tomography
1.3.1.5 Histological and immunohistochemistry staining
1.3.2 Challenges in collagen visualization
1.4 Conclusion
2 Theoretical background of polarization-resolved SHG microscopy 
2.1 Principles and contrast mechanisms of multiphoton microscopy
2.1.1 Making intense fields
2.1.2 Optical sectioning
2.1.3 Two-photon excited fluorescence (2PEF)
2.1.4 Second and third harmonic generation
2.1.5 Coherent anti-Stokes Raman Scattering
2.2 Second Harmonic Generation microscopy
2.2.1 Mechanisms and principles
2.2.1.1 Physical origins
2.2.1.2 The role of coherence in SHG
2.2.1.3 Resolution: comparison between SHG microscopy and other techniques
2.2.2 Origin of SHG in collagen
2.3 Collagen tensorial response
2.3.1 Tensorial formalism of medium polarization
2.3.2 Symmetries of collagen assemblies and nonlinear response tensors
2.3.3 Second harmonic response of collagen at molecular and fibrillar scale
2.3.4 P-SHG signal for C∞v symmetry
2.3.5 Tensorial response variation versus disorder in collagen fascicle
2.4 Conclusion
3 Linear optical effects in polarization-resolved SHG microscopy 
3.1 Experimental setup for simultaneous SHG/2PEF imaging
3.2 Experimental artefacts in thick anisotropic tissue
3.2.1 Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues
3.3 Numerical simulations
3.3.1 Overview of SHG simulation in tendon
3.3.2 Focal field calculation in birefringent media
3.3.2.1 Field propagation in a uniform medium. Angular spectrum representation
3.3.2.2 Field in a birefringent medium
3.3.2.3 Boundary conditions between isotropic and birefringent media
3.3.2.4 Results and discussion
3.3.3 SH radiation in tendon
3.3.3.1 Radiation of a punctual dipole in a birefringent medium
3.3.3.2 Radiation integral
3.3.3.3 Simplification of the calculation using relative order of magnitude and symmetry of SH radiation components
3.3.3.4 Results: angular radiation diagrams
3.3.3.5 Results: total SHG intensity polarization diagrams
3.3.3.6 Discussion of the simulated ρ and
3.3.4 SHG simulations in cornea
3.3.4.1 Introduction
3.3.4.2 In vivo structural imaging of the cornea by polarizationresolved second harmonic microscopy
3.4 Discussion
4 Tendon biomechanics 
4.1 Experimental setup
4.2 Proof of concept of biomechanical assays coupled with SHG imaging
4.2.1 Introduction
4.2.2 Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy
4.3 Varying fibril ordering in tendon
4.3.1 Introduction
4.3.2 Polarization-Resolved Second-Harmonic Generation in Tendon upon Mechanical Stretching
4.4 Discussion