In vivo, pixel-resolution mapping of thick filaments' orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy.
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.
Psilodimitrakopoulos
Sotiris
S
ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, 08860 Castelldefels, Barcelona, Spain. sotiris.psilodimitrakopoulos@icfo.es
Santos
Susana I C O
SI
Amat-Roldan
Ivan
I
Thayil
Anisha K N
AK
Artigas
David
D
Loza-Alvarez
Pablo
P
D000465:Algorithms; D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton; D008859:Microscopy, Polarization / Q000379:methods*; D032342:Muscle Cells / Q000648:ultrastructure*; D015203:Reproducibility of Results; D012518:Sarcomeres / Q000648:ultrastructure*; D012680:Sensitivity and Specificity