Abstract
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.
Highlights
Light-induced polymerization and crosslinking are widely used methods for the fabrication and processing of polymeric materials, with applications ranging from manufacturing, photolithography, and tissue engineering
While most applications rely on single-photon absorption, two-photon absorption-mediated processes using a near-infrared (NIR) femtosecond laser offers far superior spatial resolution that is confined to the focal volume
Our results demonstrate the ability to alter the microstructure and mechanical modulus locally in the cornea by 2P-CXL with microscopic resolution and illustrate the unique advantage of Brillouin microscopy in evaluating 2P-CXL induced changes nondestructively
Summary
We used a home-built, video-rate, two-photon microscope. One PMT was used for detection of second harmonic generation (400 nm ± 5 nm) and another for riboflavin fluorescence (520 nm ± 35 nm). The corneas were allowed to warm to room temperature to minimize temperature induced drift effects. The top of the corneal stroma was determined by determining the Z-plane where SHG intensity abruptly changed. To demonstrate three-dimensional, subsurface 2P-CXL, we crosslinked a vertically-aligned stack of 10 planes, equidistantly spaced 3 μm apart at depths of approximately -35 to -65 μm. The crosslinking was monitored in real time and the riboflavin fluorescence and SHG intensity were recorded at 5 second intervals. If non-negligible drift was observed during this process, crosslinking was restarted with a different sample
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