Implementation of artifact-free circular dichroism SHG imaging of collagen

International audienceSecond harmonic generation (SHG) enables in situ imaging of fibrillar collagenarchitecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopyhas been implemented to take advantage of collagen chirality to improve 3D visualization. Itmeasures the normalized difference in the SHG signal obtained upon excitation by left versusright circular polarizations. However, CD-SHG signal is not well characterized yet, and quitedifferent CD-SHG values are reported in the literature. Here, we identify two major artifactsthat may occur in CD-SHG experiments and we demonstrate that thorough optimization andcalibration of the experimental setup are required for CD-SHG imaging. Notably it requires acareful calibration of the incident circular polarizations and a perfect mechanical stabilization ofthe microscope stage. Finally, we successfully record CD-SHG images in human cornea sectionsand confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane