Purpose: Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging. Methods: Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 mu m). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 mu m) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph. Results: The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 mu m lateral, 2.0 mu m axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer). Conclusions: We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.
