Second harmonic generation (SHG) microscopy is a promising imaging technique for collagenous tissues due to its noninvasiveness and potential for 3D imaging. In tissues with densely packed and thin collagen fibrils, such as cartilage, the focal volume of the laser can comprise multiple fibrils, and the SHG from each of these fibrils can interact and contribute to the detected signal. Coherent amplification is achieved when the fibrils are aligned and oriented in the same direction. The effect of changing the size of the focal volume, as determined by the numerical aperture (NA), is therefore dependent on the length scale at which the fibrils are aligned. This effect on the image contrast is important to examine experimentally before SHG can be used in the clinic. In this study, we measured the SHG intensity and radiation direction as a function of the NA of the excitation and collecting objectives in different areas of the immature articular cartilage of young pigs. The results showed that varying the NA of the excitation objective had more effect on the SHG signals detected in the forward compared to the backward direction and that this effect varied considerably throughout the tissue. The results clearly demonstrated that SHG imaging differs from conventional histology, and the image contrast should only be interpreted in light of the imaging conditions.

Date Published: 2 March 2017
PDF: 8 pages
Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100692I (2 March 2017); doi: 10.1117/12.2249985

Published in SPIE Proceedings Vol. 10069:
Multiphoton Microscopy in the Biomedical Sciences XVII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)