In situ multiphoton microscopy for monitoring femtosecond laser eye surgery

Abstract : Purpose: The authors present a multiphoton microscope set–up mounted on a microsurgery experimental system using a Nd:glass femtosecond laser. The system allows for non–linear optical imaging before, during, and after the surgical intervention. Methods: The femtosecond laser is a CPA system with a regenerative amplifier delivering pulses at a wavelength of 1.06 µm, pulse durations of typically 400 fs and a maximum energy of 60 µJ. The repetition rate is adjustable from single shot up to 10 kHz. The delivery system provides spot sizes down to the micron range and peak power densities well above 1015W/cm2. The human corneas are mounted on an anterior chamber system, which is installed on a computer controlled step motor positioning system. Photons generated by non–linear processes in the cornea travel backwards through the front part of the beam delivery optics and are captured by a photomultiplier tube behind a dichroic mirror. The signal is filtered by a lock–in amplifier tuned to the laser repetition rate and recorded by a personal computer. Scanning the sample permits the acquisition of 2D or 3D images. Results: When working above the ablation threshold, the set–up permits to induce laser cuts following complex geometries in human cornea. Penetrating and lamellar cuts could be performed. Below the ablation threshold the femtosecond laser pulses create secondary photons by the stimulation of non–linear processes in the samples. The interaction process could be identified as being predominantly second harmonic generation (SHG) and, to a lesser extent, two–photon fluorescence. The images obtained from the multi–photon module permitted to monitor, control and optimise the surgical intervention in situ without the need to move the sample or to match results from different characterisation methods. Conclusions: The combination of multiphoton imaging and corneal surgery necessitates only minimal modifications of the optical system of a femtosecond surgical laser system which do not compromise its performance. It is demonstrated that a combined system significantly improves parameter control and the monitoring of the surgical intervention.