Fibrosis pp 409-425 | Cite as

Probing Collagen Organization: Practical Guide for Second-Harmonic Generation (SHG) Imaging

  • Riccardo CicchiEmail author
  • Francesco S. Pavone
Part of the Methods in Molecular Biology book series (MIMB, volume 1627)


Second-harmonic generation (SHG) microscopy is a powerful microscopy technique for imaging collagen and other biological molecules using a label-free approach. SHG microscopy offers the advantages of a nonlinear imaging modality together with those ones of a coherent technique. These features make SHG microscopy the ideal tool for imaging collagen at high resolution and for characterizing its organization at various hierarchical levels. Considering that collagen organization plays a crucial role in fibrosis and in its development, it would be beneficial for the researcher working in the field of fibrosis to have a manual listing crucial points to be considered when imaging collagen using SHG microscopy. This chapter provides an answer to this demand with state-of-the-art protocols, methods, and laboratory tips related to SHG microscopy. We also discuss advantages and limitations of the use of SHG for studying fibrosis.

Key words

SHG microscopy Collagen Polarization scanning Forward backward Detection Numerical aperture Ti:sapphire laser 



We acknowledge funding from the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX, from Tuscany Region and EU FP7 BiophotonicsPlus projects “LITE” (Laser Imaging of The Eye) and “LighTPatcH” (Led Technology in Photo Haemostasis), from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number 284464 and number 604102 (Human Brain Project), from the Italian Ministry of Health (GR-2011-02349626), from Fondazione Pisa, and from Ente Cassa di Risparmio di Firenze. Part of this work was performed in the frame of the Proof of Concept Studies for the ESFRI research infrastructure project Euro-BioImaging at the PCS facility LENS.


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Authors and Affiliations

  1. 1.National Institute of OpticsNational Research Council (INO-CNR)Sesto FiorentinoItaly
  2. 2.European Laboratory for Non-linear Spectroscopy (LENS)University of FlorenceSesto FiorentinoItaly
  3. 3.Department of PhysicsUniversity of FlorenceSesto FiorentinoItaly

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