High-resolution high-contrast in vivo phase imaging of corneal nerves and immune cells in mice

초록

Purpose Interactions between corneal nerves and immune cells are essential for corneal homeostasis and inflammation regulation. Although simultaneous imaging has been demonstrated, existing in vivo techniques often provide limited contrast for resolving fine cellular morphology. This study evaluates a label-free, high-resolution phase contrast microscopy approach for simultaneous visualization of corneal nerves and immune cells in mouse model. Methods A high-resolution differential phase contrast (DPC) microscopy system was developed using highly angled oblique illumination and a high numerical aperture objective. The system was applied to mouse corneas (n = 5) before and 6 h after partial circular corneal nerve cut (CNC). Three-dimensional and time-lapse imaging were performed and compared with in vivo confocal microscopy (IVCM). Immunofluorescence (IF) imaging of ex vivo corneas provided structural validation. Immune cell density in the central cornea was quantified before and after injury. Results DPC microscopy enabled clear visualization of the sub-basal nerve plexus (SBNP) and cellular structures in naïve corneas. Compared with IVCM, DPC provided more continuous visualization of nerve fibers and revealed fine immune cell structures based on refractive index variation. Following CNC, reduced visibility of SBNP fibers and an increase in cell density were observed, from 50 ± 18 to 226 ± 59 cells/mm2. Time-lapse DPC imaging enabled distinction of cells with intracellular activity. Structural features observed with DPC imaging were corroborated by IF imaging. Conclusions High-resolution DPC microscopy enables label-free visualization of corneal nerves and immune cells with improved structural continuity compared with conventional IVCM. This approach provides a versatile platform for studying corneal neuro-immune interactions, with further development, may support translational research and clinical diagnostics.

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