Comparison of second harmonic microscopy images of collagen-based ocular tissues with 800 and 1045 nm

Second harmonic generation (SHG) imaging is a well-suited multiphoton technique allowing visualization of biological tissues mainly composed of collagen with submicron resolution. Despite its inherent confocal properties, imaging of deeper layers within thick samples has still some limitations. Although the use of longer wavelengths might help to overcome this, the dependence between SHG signals and wavelength is still under discussion. We report here on the dependence with wavelength of SHG signals from collagen-based ocular tissues. The quality of SHG images for two commonly used excitation wavelengths (800 and 1045 nm) is studied. The analysis of the collagen structural information reveals that the information provided by both wavelengths is similar. It was also found that, independently of the depth location, 1045-nm SHG images presented always lower signal levels than those acquired with 800 nm. However, the contrast of the former images was higher, what may improve the visualization of certain features of interest.