Individual Astrocyte Morphology in the Collagenous Lamina Cribrosa Revealed by Multicolor DiOlistic Labeling

Abstract

Purpose: To evaluate multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous lamina cribrosa (LC). Methods: Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150μm-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. Results: DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness. Conclusions: MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

Instant polarized light microscopy pi (IPOLπ) for quantitative imaging of collagen architecture and dynamics in ocular tissues

“…Fourth, the colorful light of IPOLπ contains information on all birefringent tissue components, including various types of collagens and non-collagenous components, such as elastin and neural tissue microtubules. (Waxman et al, 2022) Since the birefringence of collagen is substantially larger than that of other birefringent elements in the eye (Inoué and Oldenbourg, 1998; Waxman et al, 2021) we assumed that the majority of the polarized light interaction was from collagen. In addition, we acknowledge that IPOLπ cannot distinguish among various collagen types.…”