Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy

Abstract

Two-photon polymerization has enabled the precise microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, and cellular scaffolds. We present two-photon collagen crosslinking (2P-CXL) of intact corneal tissue using riboflavin and femtosecond laser irradiation. The collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. The measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without the perturbation of the surrounding non-irradiated regions. 2P-CXL-induced stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering. (C) 2016 Optical Society of America