Imaging corneal crosslinking by autofluorescence 2-photon microscopy, second harmonic generation, and fluorescence lifetime measurements

PURPOSE: To evaluate the use of 2-photon microscopy (TPM), which excites tissue autofluorescence, in detecting and calculating the grade of collagen corneal crosslinks, which are not visible through the slitlamp and in vivo confocal microscopy. SETTING: Departments of Ophthalmology, University of Lubeck, Lubeck, and University of Rostock, Rostock, Germany. DESIGN: Experimental study. METHODS: Corneas of rabbits were treated with different crosslinking (CXL) protocols. Two weeks after treatment, the corneas were evaluated in vivo by confocal microscopy. Eyes were enucleated and TPM was performed at 710 nm and 826 nm excitation wavelengths to detect tissue autofluorescence, second harmonic generation, and fluorescence lifetime measurements (FLIM). Eyes were then fixed and analyzed by histology. RESULTS: Cross linking following the standard protocol generated a strong autofluorescence signal in the stroma that was detected by TPM. This signal was weakly present in the control specimens, and a sharp transition zone between the peripheral zone and the CXL zone was seen. On FLIM, an increase in corneal crosslinks was measured when the standard protocol was used. CONCLUSIONS: Two-photon microscopy, a noninvasive method, was able to detect the effects of therapeutic CXL and measure the grade of CXL. In addition to postoperative treatment control, the technique has possibilities for use in online dosimetry during 2-photon triggered CXL.