Figure 4.

Mouse eye anatomy imaged by simultaneous 2PAF and SHG. 2PM imaging was performed in an unfixed, enucleated C57BL/6 mouse eye imaged with the cornea oriented perpendicularly to the objective lens. (A) A 5 × 5 composite of tiled 2PM images (tiles each 450 μm/side) illustrates a transverse optical slice completely through the mouse eye. Red: 2PAF; blue: SHG. The iris (IR) is apparent by 2PAF signal from the melanin, and the cornea and sclera are apparent by their SHG signal. Two small regions (B, C, insets) centered on the iridocorneal angle of this eye are shown at higher resolution in (B) and (C), respectively. (B) SC is identified by the absence of an SHG signal and as correlated to its anatomic location to the IR (red). Collector channels (CCs) are visible as open structures within the scleral tissue (blue). (C) The IR is visible by the 2PAF signal (red) of the melanin contained within, the corneal is visible as the SHG signal (blue) at the top of the panel, and the SC is located as an area lacking SHG signal near the insertion point of the IR. The thin mouse trabecular meshwork (TM) layer was identified as a hazy strip of SHG signal interior to SC. (D) Hematoxylin and eosin–stained histologic section from the same region shown in C. The relative locations of the SC, TM, and IR in this histologic section are in close approximation to their locations in the 2PM image. Scale bars, 100 μm.