Specimens were examined by inverted microscope (Axiovert 200; Carl Zeiss Meditec, GmbH, Jena, Germany, equipped with a 40× [numerical aperture, 1.2] water-immersion objective lens, and having a working distance of 190 μm). Two-photon second harmonic signals from collagen were generated with a mode-locked titanium:sapphire laser (Maitai; Spectra-Physics Lasers Division, Mountain View, CA). The optimal wavelength for the generation of second harmonic signals from human corneal collagen has been found to be 800 nm.
10 Forwardscatter signals or transmitted signals that passed through the tissue were collected with the use of a condenser lens (numerical aperture, 0.55) and a narrow bandpass filter (400/50) positioned in front of the transmission light detector. Backscatter signals were collected by the microscope objective and detected (LSM 510 META detector; Carl Zeiss Meditec, GmbH) over wavelengths from 377 to 430 nm. With the multitrack mode of the detector, we obtained sequential, en face second harmonic and single-photon fluorescence signals from the same optical slice. All samples were scanned with a 1-μm step size in the
z-axis to generate 3D data sets extending from the surface of Bowman’s layer to a depth of 150 μm into the anterior stroma. The fluorescence signals from Alexa Fluor 488–phalloidin and fluorescent red nucleic acid stain (Syto 59; Molecular Probes) were excited by the 488- and 633-nm laser lines of the argon and red helium-neon lasers, respectively, and were collected by bandpass filters of 500 to 550 nm and of 650 to 710 nm, respectively. Twelve-bit 512 × 512 images were recorded. The 3D data sets were reconstructed with the use of image software (LSM Image Examiner; Carl Zeiss Meditec, GmbH). A minimum of three 3D data sets was collected from different randomly selected regions of each corneal block.