In situ multiphoton microscopy for monitoring femtosecond laser eye surgery in the human cornea and sclera

We present a multiphoton imaging system mounted on a microsurgery experimental set-up using a Nd:glass femtosecond laser. The system permits to induce laser incisions in human cornea and sclera and to perform nonlinear imaging during the intervention. The laser is a chirped pulse amplification (CPA) system with a regenerative amplifier delivering pulses at a wavelength of 1.06 μm. pulse durations of 400 fs and a maximum energy of 60 μJ at repetition rates up to 10 kHz. The delivery system provides spot sizes down to the micron range. The samples are human corneas retracted from the transplant circuit mounted on a moveable anterior chamber system. Photons generated by non-linear processes in the cornea travel backwards through 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. Scanning the sample permits the acquisition of three-dimensional microscopic images. Above the incision threshold the set-up permits to induce laser cuts in human cornea following complex geometries. Below the threshold the laser pulses create secondary photons by the stimulation of non-linear optical processes in the samples which could be identified as being predominantly second harmonic generation (SHG). The in situ images obtained from the multi-photon module permit to control and optimise the surgical intervention. The combination of multiphoton imaging and corneal surgery necessitates only minimal modifications of the optical system of a femtosecond surgical laser system. A combined system significantly improves parameter control and permits the monitoring of the surgical procedure.