Birefringence and Second Harmonic Generation on Tendon Collagen Following Red Linearly Polarized Laser Irradiation
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Birefringence and Second Harmonic Generation on Tendon Collagen Following Red Linearly Polarized Laser Irradiation DANIELA FA´TIMA TEIXEIRA SILVA,1,4 ANDERSON STEVENS LEONIDAS GOMES,2 BENEDICTO DE CAMPOS VIDAL,3 and MARTHA SIMO˜ES RIBEIRO4 1
Post-Graduation Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho (UNINOVE), Sa˜o Paulo, SP 01504-001, Brazil; 2Departamento de Fı´ sica, CCEN-UFPE, Recife, PE 50670-901, Brazil; 3Instituto de Biologia, IB-UNICAMP, Campinas, SP 13084-971, Brazil; and 4Centro de Lasers e Aplicac¸o˜es, IPEN-CNEN/SP, Sa˜o Paulo, SP 05508-900, Brazil (Received 15 May 2012; accepted 5 December 2012; published online 18 December 2012) Associate Editor James Tunnell oversaw the review of this article.
certain extent it is responsible for the physical properties of connective tissue. Type I collagen, found in the skin, tendons, bones and other connective tissues of vertebrate animals, is one of the most abundant proteins in the living organism.19 In tendons, where collagen concentration may reach 80–90% of the dry mass, collagen fibers can be linked directly to the biomechanical function of transmitting forces from the muscle to the bone.13 Collagen molecules have nanometrical dimensions, are around 300 nm in length and 1.43 nm in diameter and build superstructures, such as tendons, by means of self-assembly, and in accordance with supra-molecular chemistry laws.8 The high degree of longitudinality of the fibrils, which extend to the organizational level of the bundles, is responsible for optical anisotropies, such as birefringence and nonlinear susceptibility.12,29 Collagen birefringence can be studied with the use of polarized light microscopy. In brief, the brightness of a birefringent object is due to refraction index difference Dn = (ne 2 no). The phase shift is d ¼ 2p k LDn, where k is the wavelength (lm), L is the sample thickness (lm), Dn is the birefringence. LDn is the optical retardation. The collagen fiber, when placed between two polarizers, as occurs under a polarization microscope, shows maximum brightness when one of its axes of propagation is placed at an angle of 45 in relation to the polarizer. Thus, the polarized light emerges from the collagen in two wave fronts, after crossing the collagen. These two fronts represent a phase difference, which is related to the optical retardation. Bigger phase difference and greater optical retardation means a more organized structure around the ordinary and extraordinary axes.12
Abstract—Regarding the importance of type I collagen in understanding the mechanical properties of a range of tissues, there is still a gap in our knowledge of how proteins perform such work. There is consensus in literature that the mechanical characteristics of a tissue are primarily determined by the organization of its molecules. The purpose of this study was to characterize the organization of non-irradiated and irradiated type I collagen. Irradiation was performed with a linearly polarized HeNe laser (k = 632.8 nm) and characterizatio
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