Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively.
Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue 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.
Sheldon J. J. Kwok, Ivan A. Kuznetsov, Moonseok Kim, Myunghwan Choi, Giuliano Scarcelli, and Seok-Hyun Yun, "Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy (Conference Presentation)," Proc. SPIE 10067, Optical Elastography and Tissue Biomechanics IV, 100670M (Presented at SPIE BiOS: January 29, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2251565.5380018832001.
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