In vivo, pixel-resolution mapping of thick filaments' orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy

J Biomed Opt. 2009 Jan-Feb;14(1):014001. doi: 10.1117/1.3059627.

Abstract

The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Animals
  • Caenorhabditis elegans / cytology
  • Image Enhancement / methods*
  • Image Interpretation, Computer-Assisted / methods*
  • Imaging, Three-Dimensional / methods*
  • Microscopy, Electron, Scanning / methods
  • Microscopy, Fluorescence, Multiphoton
  • Microscopy, Polarization / methods*
  • Muscle Cells / ultrastructure*
  • Reproducibility of Results
  • Sarcomeres / ultrastructure*
  • Sensitivity and Specificity