Investigation of Collagen Crosslinks Introduced By a Femtosecond Laser

The treatment of the growing number of short-sighted individuals worldwide [1], as well as the treatment of keratoconus-a severe corneal disease-requires precise procedures for reshaping the cornea in cases of myopia or increasing corneal stiffness in cases of keratoconus. We have previously shown that direct crosslinking of corneal collagen fibres can be achieved using fs laser pulses at λ = 258 nm without using additional sensitizers [2,3]. Thereby, stiffness can be modulated locally which opens the door to individually shaped corneas [4]. Enhancement of autofluorescence is a well-known side effect of collagen crosslinking when using UV light with chemical sensitizers or sensitizers alone [5,6] and is also present when using UV fs laser pulses alone. Here, we characterized the autofluorescence using spectral imaging and fluorescence lifetime imaging microscopy (FLIM). Gelatine samples as simplified collagen model were swollen up in distilled water and then treated with fs laser pulses at λ = 258 nm provided by twofold frequency conversion of a λ = 1030 nm laser pulse line. The fluorescence spots generated by fs-laser treatment were examined using a Zeiss CLSM780 Meta microscope for spectral and a Zeiss CLSM980 microscope with an attached rapidFLIM™ module (Picoquant) for FLIM measurements.