Open AccessImplementation of artifact-free circular dichroism SHG imaging of collagen
Author(s) -
Margaux Schmeltz,
Claire Teulon,
Gaël Latour,
Djida Ghoubay,
Vincent Borderie,
Carole Aimé,
Marie-Claire Schanne-Klein
Publication year - 2019
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.27.022685
Subject(s) - second harmonic generation , optics , materials science , second harmonic imaging microscopy , circular dichroism , microscopy , microscope , chemistry , physics , crystallography , laser
Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane.