Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

Abstract

The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures.

Keywords: (170.1020) Ablation of tissue; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology; (180.4315) Nonlinear microscopy.

Fig. 1

(a) Transversal histological section of a chicken cornea. (b)-(f) TPEF images of the different layers within the epithelium: superficial cells (b), wing cells (c-d), basal cells (e-f). The three types of epithelial cells can be observed together in (g) (see stars). Bar length: 50 μm.

Fig. 2

SHG signal from the collagen of the chicken corneal stroma at different depths (spaced ~30 µm). Bar length: 50 μm.

Fig. 3

Histological sections of chicken corneas showing the intrastromal ablation patterns #1 (a) and #2 (b). Bar length: 200 μm.

Fig. 4

Microscopy imaging illustrating the intrastromal laser surgery: bright-field (a, c) and nonlinear images (b, d). Upper and bottom rows correspond to ablation patterns #1 and #2 respectively. Each square sets the size of the adjacent image. The size of images (b) and (d) is 840x840 μm².

Fig. 5

3D representation of the intrastromal ablated area. 20 individual SHG images of planes 2-μm apart were used for the reconstruction. Blue color has been used for a better visualization of the cavities of pattern #1. Image size corresponds to the square inset in Fig. 3(b).

Fig. 6

3D (nonlinear microscopy) images of chicken corneas showing the two different ablation patterns: (a) #1 and (b) #2.

Fig. 7

Nonlinear microscopy images of an ablated chicken cornea (pattern #1) at different depths (spaced 40 μm). The imaged area was 840x840 μm².

Fig. 8

(Media 1) Movie showing the stack of nonlinear images at different depths in an ablated cornea.

Fig. 9

Nonlinear microscopy images of an ablated chicken cornea (pattern #2) corresponding to planes 30 μm apart. Image dimensions are the same as for Fig. 6.

Fig. 10

(a) Examples of locations across the nonlinear microscopy image of an ablated chicken cornea (pattern #1) where the intensity profile was computed. (b) Intensity along two different sections crossing the location of three cavities of the intrastromal pattern.