Using a fluorescence microscopic camera, we observed pooling of the fluorescein solution in the central cornea (
Figs. 1B,
1C) after the stromal injection. The injected dye appeared as ring-shaped fluorescence due to the concentration quenching of fluorescein.
18,19 In normal corneas, the water began to flow from the point of central pooling to the corneal periphery in a linear fashion (
Fig. 2A) 3 minutes after the injection. Upon reaching the peripheral cornea (
Fig. 2B) 4 minutes after the injection, the water swirled in an arc along the most peripheral area (
Fig. 2C) 5 minutes after the injection, and the range of the fluorescence flow (
Fig. 2D) expanded 8 minutes after the injection and ultimately covered the entire cornea (
Figs. 2E,
2F) 15 and 20 minutes, respectively, after the injection. These results indicated the presence of horizontal water migration in the entire normal cornea. Although we observed slow centrifugal diffusion of the fluorescein from the point of injection in all directions to the peripheral cornea, we primarily observed a rapid unidirectional spiral pattern of fluorescein originating from the point of injection. The spiral pattern was driven by in vivo intracorneal water migration. The values of IOP measured by a tonometer (Icare Finland Oy, Vantaa, Finland) before the experiment (average ± SEM, 7.8 ± 0.5 mm Hg;
n = 5) did not differ significantly from those measured after the experiment (8.5 ± 0.8 mm Hg). The measurements of the fluorescent area and estimations of their extent over time as a percentage of the entire corneal area were 3.4% ± 0.2%, 7.8% ± 1.1%, 19.6% ± 3.2%, 52.9% ± 12.6%, 80.1% ± 6.3%, and 91.8% ± 4.8% (average ± SEM;
n = 5) at 0, 3, 5, 8, 15, and 20 minutes, respectively. Analysis of the expansion of the flow of water (
Fig. 2G) indicated that the fluorescent-positive area gradually increased and encompassed the entire cornea throughout the time of observation.
Supplementary Video S1 is a time-lapse video of the normal cornea after intrastromal injection of the fluorescein solution. The video shows the details of the dynamic appearance of the swirling flow of water.