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

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.

Fig. 1

Home-built two-photon microscope. The cornea sample was compressed with a coverslip. SHG (blue, 400–410 nm) and fluorescence (green, 485–555 nm) was collected through appropriate dichroic mirrors and filters to separate photomultiplier tubes (PMTs).

Fig. 2

Assessment of one-photon CXL with multiphoton imaging. (a) Reconstructed depth profile of riboflavin soaked corneas before and after CXL. Green: riboflavin fluorescence, Blue: backscattered collagen SHG. (b) Summary of changes, with mean ± std. error are reported. *, p<0.05. (c) Top: distribution of collagen fiber orientations determined by 2D Fourier transform. Red curve indicates fitting to von Mises distribution. Bottom: standard deviation (σ) of the fiber orientation. A uniform fiber distribution is defined to have a σ of 0.5 radians (dotted line). ****, p<0.0001

Fig. 3

Determination of laser parameters for 2P-CXL. (a) Two-photon bleaching time constant (1/e) in seconds as a function of incident laser power (mW). Mean ± std. error are reported. Black line is linear fit to ln(τ) vs. ln(power). (b) Changes in SHG and fluorescence with 2P-CXL. Mean (solid line) ± std. error (dotted line) are reported. Inset: change in the standard deviation (σ) of fiber orientations. (c) At 207 mW, there is no discernable tissue damage with 20 min irradiation, while at 312 mW, there is noticeable photodamage (white arrow) after 5 min. SHG (blue) and fluorescence (green) channels are shown. Scale bar: 50 μm.

Fig. 4

Brillouin microscopy. (a) Brillouin scattering is due to interaction of photons and spontaneous acoustic waves. For the 2P-CXL region, the acoustic velocity is higher while the phonon wavelength is assumed to be constant. (b) Confocal Brillouin microscope connected to a two-stage, apodized virtually imaged phased array (VIPA) spectrometer. A 0.3 NA objective lens was used.

Fig. 5

Brillouin microscopy of a 2P-CXL-treated cornea. (a) x-y images of the 2P-CXL region, at 15, 45, 75, and 105 μm below the cornea surface. Scale bar: 50 μm. b) x-z image, where top refers to the surface of the cornea. (c) Three-dimensional view of the 2P-CXL region reconstructed and interpolated from thirteen x-y images (selected images shown in a) at 10 μm intervals in z.

Fig. 6

Quantification of 2P-CXL-induced stiffening. (a) Mean (± stdev.) Brillouin shift of the crosslinked and non-irradiated regions of the cornea as a function of depth. (b) Relative Brillouin shift obtained from subtracting the profiles in (a) fit to a Gaussian function (R² = 0.99).