Estimation of helical angles of myosin and collagen by second harmonic generation imaging microscopy

François Tiaho,Denis Rouède,Gaëlle Recher

doi:10.1364/oe.15.012286

Abstract

We performed Second Harmonic Generation (SHG) imaging microscopy of endogeneous myosin-rich and collagen-rich tissues in amphibian and mammals. We determined the relative components of the macroscopic susceptibility tensor chi((2)) from polarization dependence of SHG intensity. The effective orientation angle theta(e) of the harmonophores has been determined for each protein. For myosin we found theta(e) approximately 62 degrees and this value was unchanged during myofibrillogenesis. It was also independent of the animal species (xenopus, dog and human). For collagen we found theta(e) approximately 49 degrees for both type I- and type III- rich tissues. From these results we localized the source of SHG along the single helix of both myosin and collagen.

Highlights

Second harmonic generation (SHG) imaging microscopy has recently appeared to be a powerful tool to image intrinsic subcellular signals from endogeneous proteins such as microtubule, myosin and collagen in living tissues [1, 2, 3]
In order to provide more information on the origin of SHG in fibrilar proteins, we investigated in the present work the polarization dependence of SHG intensity generated by thin slices of striated muscles and of collagen-rich tissues from different species (Xenopus laevis, dog and human)
To determine the effect of myofibrillogenesis on SHG signal, we have studied xenopus tadpoles swimming muscles from 1, 2 and 4 days post fertilization

Summary

Introduction

Second harmonic generation (SHG) imaging microscopy has recently appeared to be a powerful tool to image intrinsic subcellular signals from endogeneous proteins such as microtubule, myosin and collagen in living tissues [1, 2, 3]. In order to provide more information on the origin of SHG in fibrilar proteins, we investigated in the present work the polarization dependence of SHG intensity generated by thin slices of striated muscles and of collagen-rich tissues from different species (Xenopus laevis, dog and human). For all myosin-rich tissues, we found that this angle was almost constant with a mean value of about θe ≈ 62◦ This value was independent of (i) the muscle developmental stage for Xenopus laevis and (ii) the studied species. In collagen I- and III-rich tissues, we found a distinct mean angle of about θe ≈ 49◦ These values are close approximations of the helical pitch angle deduced from X-rays structural parameters of helices of both myosin and collagen [11]

Methods

Results

Discussion

Conclusion