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Two-photon polymerization has enabled 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. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Measurement of local changes in longitudinal mechanical moduli with confocal Brillouin microscopy enabled the visualization of the cross-linked pattern without 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 resolution with broad implications in ophthalmology, laser surgery, and tissue engineering.

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