Chapters authored
Second Harmonic Generation Microscopy: A Tool for Quantitative Analysis of Tissues
Second harmonic generation (SHG) is a second‐order non‐linear optical process produced in birefringent crystals or in biological tissues with non‐centrosymmetric structure such as collagen or microtubules structures. SHG signal originates from two excitation photons which interact with the material and are “reconverted” to form a new emitted photon with half of wavelength. Although theoretically predicted by Maria Göpert‐Mayer in 1930s, the experimental SHG demonstration arrived with the invention of the laser in the 1960s. SHG was first obtained in ruby by using a high excitation oscillator. After that starting point, the harmonic generation reached an increasing interest and importance, based on its applications to characterize biological tissues using multiphoton microscopes. In particular, collagen has been one of the most often analyzed structures since it provides an efficient SHG signal. In late 1970s, it was discovered that SHG signal took place in three‐dimensional optical interaction at the focal point of a microscope objective with high numerical aperture. This finding allowed researchers to develop microscopes with 3D submicron resolution and an in depth analysis of biological specimens. Since SHG is a polarization‐sensitive non‐linear optical process, the implementation of polarization into multiphoton microscopes has allowed the study of both molecular architecture and fibrilar distribution of type‐I collagen fibers. The analysis of collagen‐based structures is particularly interesting since they represent 80% of the connective tissue of the human body. On the other hand, more recent techniques such as pulse compression of laser pulses or adaptive optics have been applied to SHG microscopy in order to improve the visualization of features. The combination of these techniques permit the reduction of the laser power required to produce efficient SHG signal and therefore photo‐toxicity and photo‐damage are avoided (critical parameters in biomedical applications). Some pathologies such as cancer or fibrosis are related to collagen disorders. These are thought to appear at molecular scale before the micrometric structure is affected. In this sense, SHG imaging has emerged as a powerful tool in biomedicine and it might serve as a non‐invasive early diagnosis technique.
Part of the book: Microscopy and Analysis