On the Relationship between Forward and Backward Second-Harmonic Generation in Full-Field Images of the Lamina Cribrosa.

Abstract

Purpose : The complex network of collagen fibrils in the lamina cribrosa provides mechanical support for neural signal pathways to the visual cortex. It can be visualized optically with wide field polarimetric imaging or with background-free second-harmonic generation when irradiated by intense fs-laser pulses. Here, we make a direct comparison between the forward and backward second-harmonic generation images captured to gain insight into the optical contrast mechanisms involved in both modalities. Ultimately, this will be required for in-vivo imaging.

Methods : The lamina cribrosa of normal sheep eyes, prepared into 30 µm coronal sections under zero mmHg intraocular pressure (IOP), has been imaged in the forward and backward directions using second-harmonic generation microscopy from a fs-laser laser (Micra Coherent^TM) and galvanometric mirror scanners. Two photomultipliers (SensTech^TM) capture simultaneous images of forward and backward second-harmonic generation. The entire cross-section of the lamina cribrosa about 4mm width is analyzed using a motorized XY sample stage (Thorlabs^TM) to facilitate stitching of the high-resolution images captured with a dry objective of NA=0.95. Circular polarization was used to prevent direction-dependent fibrillar visualization. We determine fibrillar linearity and orientation across the full width of the laminar cribrosa using the Hough Transform (Matlab^TM).

Results : Images captured in the forward and backward directions show delicate differences related to the phase relations of the coherent second-harmonic signal. The linearity of fibrils in the fixed sample cross sections has been evaluated and is found to be approximately an order-of-magnitude higher in the forward direction. The results are presented for the Hough Transform for both forward and backward second-harmonic generation images that confirm our findings.

Conclusions : We find that second-harmonic generation imaging produces the highest linearity and matches structure in the forward direction, whereas in the backward direction images provide complementary insight into how the fibrils scatter collectively. Fibrillar linearity may break down in case of prolonged elevated intraocular pressure but for the samples studied is relatively low. The second-harmonic signal provides enhanced optical resolution and contrast.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.