Polarimetric response of second harmonic generation in microscopy of chiral fibrillar structures

Abstract

Polarimetric second harmonic generation (SHG) microscopy is employed to study partially oriented fibrillar structures. The polarimetric SHG parameters are influenced by three-dimensional (3D) configuration of C6 symmetry fibrilar structures in the focal volume (voxel) of a microscope. The achiral and chiral susceptibility tensor components ratios (R and C, respectively) are extracted from the linear polarization-in polarization-out (PIPO) measurements. The analytical derivations along with the polarimetric SHG microscopy results obtained from rat tail tendon, rabbit cornea, pig cartilage and meso-tetra (4-sulfonatophenyl) porphine (TPPS4) cylindrical aggregates demonstrate that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and R and C ratio values change by tilting the fibers out of image plane, as well as by crossing the fibers in 2D and 3D. The polarimetric microscopy results are consistent with the digital microscopy modeling of fibrillar structures. These results facilitate the interpretation of polarimetric SHG microscopy images in terms of 3D organization of fibrilar structures in each voxel of the samples.

Determining the helical pitch and relative polarity of collagen using polarization-resolved SHG

“…its sign is different between two sides of the same buckle) as expected based on equation (2b). The CD values are found to be almost always positive near the center of the fibril, where we know polarity changes are occurring, indicating that CDSHG reveals out of plane fibrils, but not their relative polarity, in agreement with [5] and contradicting [7,12]. Some regions near the edge of the fibril are found to have negative CD values which we attribute to an edge effect resulting from the nonuniform focal field as is the case for ρ.…”

“…preference for right-handed vs left-handed excitation) reveals the relative out of plane polarity of collagen fibrils [6][7][8][9][10][11]. However recent attempts to test this hypothesis on collagen in tissue have yielded contradictory results [5,7,12]. Results from individual collagen fibrils are therefore desirable and will provide clarification on the ability of PSHG to provide three dimensional images of collagen organization within tissue.…”