Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study

Mehdi Alizadeh; Fayez Habach; Mykolas Maciulis; Lukas Kontenis; Saulius Bagdonas; Serguei Krouglov; Vytautas Baranauskas; Danute Bulotiene; Vitalijus Karabanovas; Ricardas Rotomskis; Margarete K Akens; Virginijus Barzda

doi:10.1016/j.bpj.2023.08.015

Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study

Biophys J. 2023 Oct 3;122(19):3937-3949. doi: 10.1016/j.bpj.2023.08.015. Epub 2023 Aug 23.

Affiliations

¹ Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada; Department of Physics, University of Toronto, Toronto, Ontario, Canada; Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania.
² Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada.
³ Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania.
⁴ Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania; Light Conversion, Vilnius, Lithuania.
⁵ Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada; Department of Physics, University of Toronto, Toronto, Ontario, Canada.
⁶ Institute of Biochemistry, Life Science Center, Vilnius University, Vilnius, Lithuania.
⁷ Biomedical Physics Laboratory, National Cancer Institute, Vilnius, Lithuania.
⁸ Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania; Biomedical Physics Laboratory, National Cancer Institute, Vilnius, Lithuania; Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius, Lithuania.
⁹ Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania; Biomedical Physics Laboratory, National Cancer Institute, Vilnius, Lithuania.
¹⁰ Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; Techna Institute, University Health Network, Toronto, Ontario, Canada; Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
¹¹ Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada; Department of Physics, University of Toronto, Toronto, Ontario, Canada; Laser Research Centre, Faculty of Physics, Vilnius University, Vilnius, Lithuania. Electronic address: virgis.barzda@utoronto.ca.

PMID: 37621088
PMCID: PMC10560685 (available on 2024-10-03)
DOI: 10.1016/j.bpj.2023.08.015

Abstract

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS₄) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C₆ symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS₄ aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.