High-resolution dual-modality imaging for collagen fibers and blood vessels in lamina cribrosa (LC) beams

Abstract

Purpose : The LC collagen fibers and vessels have different functions. They are also heavily intertwined, suggesting some interplay in their mechanics (PMID: 33001158). We developed a high-resolution dual-modality microscope to measure the stretch-induced LC collagen and vessel deformation.

Methods : We modified a commercial microscope (Nikon Az100M) equipped with a monochrome CMOS camera to enable instant polarized light microscopy pi (IPOLπ) and epifluorescence (Fig. 1). IPOLπ optically encodes collagen density and orientation into color, with a halogen lamp and red, green, and blue filters switched by a high-speed filter wheel to produce color images. For visualization, the brightness of IPOLπ images at each pixel was weighted using color-energy mapping. Vessels were labeled by tomato lectin, and the fluorescence imaged using a SOLA light engine with a Cy5.5 filter. A coronal section of pig optic nerve head was mounted and imaged while stretched biaxially quasi-statically. At each stretch step we measured the lengths of LC collagen and vessels.

Results : LC collagen and vessels, as narrow as 5µm, were detected using the dual-modality imaging system (Fig. 2A). During stretch testing, beam elongation was significantly larger than for vessels (Fig. 2B).

Conclusions : We demonstrated dual-modality imaging using a monochrome camera, which has higher sensitivity and spatial resolutions than a color camera. The two imaging modalities can be easily alternated through a filter cube switcher. Although the number of samples and measurements tested was small, the results suggest the intriguing finding that LC collagen stretched almost twice as much as vessels. This could be a mechanism to protect LC vessels from stretch-induced damage.

This abstract was presented at the 2023 ARVO Imaging in the Eye Conference, held in New Orleans, LA, April 21-22, 2023.

 

Dual-modality imaging was implemented on Nikon Az100M. The two imaging modalities are naturally aligned because of sharing the same imaging path.

 

(A) A dual-modality image of the pig optic nerve head for collagen and vessels (black arrow) visualization and a signal profile of a vessel (blue line). Hue represents collagen orientation; white represents vessels. (B) Two vessels (red and blue arrows) were located at LC beams. Note that the dark background is due to post-processing using IPOLπ color-energy mapping. Percentage changes in collagen length (yellow arrow) and vessel lengths with increased stretch steps.