Experimental Retinal Ablation and Imaging With Femtosecond Laser Pulses

Abstract

Abstract: : Purpose: Amplified femtosecond lasers with µJ pulse energies are novel tools in corneal surgery, but little is known about use of femtosecond pulses in retinal microsurgery. Studies indicate that nJ laser pulses of non–amplified femtosecond lasers offer better precision and less collateral tissue damage compared to mJ and µJ laser pulses of amplified femtosecond lasers. We report preliminary results of in vitro intraretinal tissue ablation with an non–amplified 90MHz femtosecond laser and high–resolution multiphoton imaging with near infrared multiphoton laser scanning microscopy. Methods: Retina from porcine and human eyes was irradiated with a laser beam derived from a compact 90 MHz modelocked Titanium:sapphire laser (1W output power, <140 fs pulse width, 720 to 950 nm wavelength tuning range, Chameleon^TM, Coherent, Santa Clara, CA). The laser was coupled to an inverted modified infrared laser scanning microscope. The system was used to perform microsurgery as well as to obtain intratissue multiphoton images before and after ablation. The laser beam was focused through a 40x oil objective (Zeiss Neofluar^TM, Zeiss, Jena, Germany) with a numeric aperture of 1.3. In addition, specimens were studied with transmission electron microscopy. Results: Line shaped intraretinal cuts were created at different wavelegths and tissue depths (15 to 30 µm). The lesion width was below 5 µm. Laser scanning microscopy revealed intratissue autofluorescence images with submicron resolution. A spectral analysis was performed. Increased fluorescence was determined at the margins of the tissue cuts, indicating small areas of collateral tissue damage. In addition, small cavitation bubbles were observed. Conclusions: Non–amplified femtosecond laser pulses at low nJ pulse energies have been applied for precise intraretinal ablation at predefined targets. High resolution multiphoton laser scanning microscopy offers functional imaging within seconds after laser ablation. The technique allows to predefine targets and to observe the progression of laser tissue removal. Our results serve as a basis for further experiments of retinal lasersurgery, e.g. removal of epiretinal membranes.

Keywords: laser • retina • microscopy: light/fluorescence/immunohistochemistry