Two-photon fluorescence imaging of riboflavin treated corneas was performed with the commercially available multiphoton microscope TriM Scope II (LaVision Biotec GmbH, Bielefeld, Germany). This microscope system was equipped with the laser system Chameleon Ultra II (Coherent, Inc., Santa Clara, CA, USA) providing fs-laser pulses in the near infrared. For imaging experiments, the artificial anterior chamber was fixed in a custom made holder beneath the objective lens (W Plan-Apochromat ×20/1.0; Zeiss GmbH, Oberkochen, Germany). This objective lens was used for focusing the laser into the corneas and is chromatically corrected for coverslips with a thickness of 0.17 mm. The coverslip separated the stromal surface from the immersion medium (water), which we used with the objective lens to reach its full numerical aperture (NA) and to prevent alteration of the diffused riboflavin in the cornea through undesired dilution by the immersion medium. The coverslip was mounted on a custom made holder (
Figs. 1b,
1c). One of the riboflavin absorption maxima is approximately 445 nm. Since scattering inside the cornea becomes smaller with increasing wavelength, a central laser wavelength of 900 nm was used for riboflavin excitation in all experiments.
10,20 For signal detection, the microscope system was equipped with two photomultiplier tubes arranged in backward direction (behind the objective lens). To restrict the detectable wavelength range, bandpass filters were used in front of every photomultiplier tube. For detecting the riboflavin fluorescence, a bandpass filter with a transmission window of 525 ± 25 nm was used. In the second detection pathway, second harmonic signals generated at the stromal collagen could be detected simultaneously when using a bandpass filter with a transmission window of 450 ± 35 nm in front of the second photomultiplier tube. A scheme of the total setup is shown in
Figure 1c. For each cornea, images were recorded in z-stacks with a step size of 10 μm over a depth of 350 μm. One single image inside the stack covered an area of 400 × 400 μm
2 with a pixel resolution of 1042 × 1042. In addition, one dark image (same settings, but without laser irradiation) was taken after every z-stack. After saturation, each cornea was imaged again in a z-stack with the same parameters as described before. The saturation process was necessary to correct our data for absorption and scattering effects.