BIOPHOTONICS: Microscopy shows corneal laser welding speeds healing

In ophthalmic surgery, laser welding of corneal tissue is an alternative to conventional suturing procedures. Although the method has been tested only at the experimental level, preliminary results show that the technique reduces inflammation, shortens surgery time, eliminates the need for suture materials that can cause irritation, speeds healing, and reduces the chance of postoperative astigmatism. However, because laser radiation absorbed directly by the water content of the cornea can cause a temperature rise that leads to collagen shrinkage, researchers in Italy have developed a laser-welding technique that uses a near-IR low-power diode laser that interacts with a chromophore solution (indocyanine green (ICG) in sterile water) to reduce temperature rise.¹ Furthermore, the research team has developed a new technique called multispectral imaging autofluorescence microscopy (MIAM) that shows the superiority of the laser-welding technique without requiring chemical manipulation of the corneal tissue.

The healing-process study was conducted by researchers from Istituto di Fisica Applicata, Università degli Studi di Firenze, and Centro di Eccellenza Optronica (CEO), all in Florence, Italy, and U.O. Oculistica in Prato, Italy. The MIAM technique, which supplies information on cell and tissue morphology and function by analyzing the spectral and spatial distribution, as well as the emission intensity of the endogenous fluorophores such as collagen, was compared directly to histological data after laser-welding surgery on 30 rabbits.

Laser welding was performed on corneal tissue treated with the ICG/water solution by an aluminum gallium arsenide diode laser emitting 80 mW at 810 nm and delivered by an optical fiber with a 300-µm core diameter. The laser treatment was applied to 5-mm-long corneal cuts performed with a standard surgical knife, and compared to identical corneal cuts left to heal naturally.

The MIAM technique used an inverted epifluorescence microscope equipped with oil-immersion objectives and with 365-nm light excitation provided by a high-pressure mercury lamp. The fluorescence signal was transmitted through a dichroic mirror and detected by a digital CCD camera. Multispectral sequential acquisition was achieved through a motorized filter wheel in front of the camera that contained different interference filters. Tissue images were acquired in three spectral bands from 430 to 470, 530 to 570, and 630 to 670 nm, corresponding to blue, green, and red fluorescence images. These monochrome images were then combined into a single color image.

Restoration begins quickly

Data from the untreated and laser-welded corneas were compared using MIAM after 7, 15, 30, 60, and 90 post-operative days. After only 7 and 15 days for the laser-welded samples, collagen fibers appeared organized in bundles and restoration of the epithelium had begun. For the untreated corneas, the epithelium and connective fibers were still completely disorganized. After 60 days, the laser-welded corneas were fully recovered, while the untreated corneas still showed irregular epithelium and wavy connective-tissue structures (see figure).

Not only was the healing process faster for the corneal tissue treated by laser welding but the organization of the stromal tissues was improved and inflammatory processes were reduced when compared to untreated corneal cuts.

The research team recognizes that autofluorescence analysis techniques like MIAM have the potential to perform real-time in vivo tissue analysis without requiring biopsies or animal sacrifice. Researchers Roberto Pini and Francesca Rossi note that laser welding of the cornea is currently leaping from the research lab to the clinic to provide sutureless closure in corneal-transplant and cataract surgery. In this respect, MIAM could become a useful diagnostic tool to monitor the healing process in patients.

REFERENCE

1. R. Pini et al., Photonics West BiOS Conference 6138, poster 6138 (January 24, 2006).