In this study, second harmonic generation signals (SHG) imaging and confocal imaging were performed on an inverted laser scanning multiphoton microscope (Zeiss LSM 510 META, Carl Zeiss, Jena, Germany) equipped with a mode-locked titanium:sapphire laser (Chameleon, Coherent, Santa Clara, CA, USA), which has a center wavelength of 800 nm for the experiments.26–28 A schematic drawing of this microscope is shown at Fig. 1. An Acoustic Optic Modulator (AOM) (Carl Ziess, Jena, Germany) was employed to attenuate the intensity of the illumination laser. The femtosecond oscillator produces a 150 femtoseconds pulse with a 90-MHz repetition rate. An illumination laser beam was circularly polarized with a quarter wave plate interposed between the oscillator and the microscope. Specimens were imaged with , , and oil-immersion objective lenses (Carl Ziess, Jena, Germany). The confocal pinhole was set to the maximal open setting for SHG application. The backward SHG signals were collected from the same objective and detected over the wavelengths from 377 to 430 nm with the detector on microscope (META detector on the model 510, Carl Ziess, Jena, Germany). Maximum input laser power was used to create the maximum SHG signal from tissue for the optimal SHG image quality. The pinhole was used for confocal application, and the reflected/backscattered laser signals were collected through the same objective and were directed by a main dichroic beam splitter to the photomultiplier tube detector (PMT2). Some samples were scanned using 5-micron vertical step size to generate 3-D data to a depth of 200 micron within the sclera. All images were recorded as 12-bit, images. 3-D data sets were reconstructed using the LSM Image Examiner (Carl Zeiss, Jena, Germany).