To determine the optimal wavelength for NLO riboflavin photoactivation, riboflavin fluorescent signal intensity as a function of femtosecond (FS) laser wavelength was measured. Riboflavin-5-phosphate (0.1%, Sigma-Aldrich, St. Louis, Missouri) in phosphate buffered saline (PBS; pH 7.3) was placed in a glass coverslip bottom Petri dish (MetTeK, Corporation, Ashland, Massachusetts) and mounted on the stage of a Zeiss, LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Jena, Germany). A 100 mW FS laser beam (Chameleon, Coherent Inc, Santa Clara, California) was then focused using a Zeiss Apochromat objective (Carl Zeiss), and the fluorescent signal was collected using the Meta Detector (Carl Zeiss) over the spectral range of 506 to 559 nm to include the peak signal (520 to 530 nm).28 To appropriately set detector gain and offset and to provide maximum intensity of signal at peak excitation, the edge of the riboflavin solution in the Petri dish well was imaged providing the simultaneous detection of riboflavin fluorescence and negative background. Initial experiments indicated that peak excitation of riboflavin was obtained with 760 nm excitation at 100 mW of laser power. Gain and offset were then adjusted to achieve a full range of pixel intensity. Images were then taken at different FS laser wavelengths from 740 to 960 nm with the laser power adjusted to 100 mW at each wavelength maintaining the same gain and offset. The intensity of signal was then averaged over a rectangular region from the region of riboflavin fluorescence and negative background using Metamorph image processing software (Molecular Devices, Sunnyvale, California).