Cell markers confirm tissue resident and infiltrating immune cells can be imaged by phase contrast adaptive optics ophthalmoscopy

Abstract

Purpose : Immune responses are associated with ocular diseases including AMD, uveitis and diabetic retinopathy and can complicate treatments including retinal gene therapy and stem cell transplantation. Directly imaging single immune cells in the living retina could elucidate the natural history of these diseases and their response to therapeutics. Here we image tissue resident and infiltrating immune cells in the mouse eye using label-free adaptive optics phase contrast ophthalmoscopy and confirm their phenotype with antibody and transgenic based markers.

Methods : An adaptive optics scanning light ophthalmoscope was used for intravital imaging of the retina in anesthetized C57BL/6J or Cx3cr1^GFP/+ mice, using phase contrast of reflected 796 nm light. Intravitreal injection of 0.5 ng lipopolysaccharide (LPS) was used to induce acute inflammation before eyes were imaged at the same retinal location from 6 hours to 2 months. Simultaneous GFP or Alexa-fluor-647 fluorescence imaging used 488 and 640 nm laser lines with 520/35 nm and 676/29 nm filters respectively.

Results : Single immune cells inside and outside of retinal vessels were visible when mouse eyes were inflamed by LPS. Following post-processing, time lapse imaging revealed highly motile cells (Fig1a) within 6 hours of injection. Cells inside post-capillary venules demonstrated leukocyte rolling and crawling without exogenous contrast (Fig1b). We confirmed most of this observed population (mean 83%, 23 cell recordings) were neutrophils by tail vein injection of Alexa Fluor-647 conjugated anti-Ly6G antibody (Fig1c). Tracking the same location (Fig1d) revealed less motility at 24 hours than 6 hours despite cellular persistence (mean cell displacement 5.9 μm vs 14.7 μm respectively, n=6 eyes, p<0.01). Microglia and hyalocytes could also be visualized label-free including their highly motile processes, confirmed using healthy eyes from Cx3cr1^GFP/+ mice (Fig1e).

Conclusions : We demonstrate longitudinal label-free immune cell imaging in vivo within the mouse retina for the first time using phase contrast adaptive optics and confirmed both tissue resident myeloid cells and infiltrating neutrophils could be visualized and tracked. Positively identifying these populations in the mouse enables the translation of this approach towards studying immune responses at the cellular level in humans.

This is a 2020 ARVO Annual Meeting abstract.

 

FIGURE 1. Scale bars = 10μm.