Development of two-photon microscopy based methods for the cornea - diagnosis of corneal infection and measurement of trans-corneal drug delivery

DoctorHigh-resolution three-dimensional (3D) imaging of the cornea is important to diagnosis corneal infection and to study corneal immune response and therapeutic effect. Confocal microscopy (CM) is widely used in the clinic to examine cornea at high-resolutions and to detect infectious pathogens based on light reflection. However, CM imaging technique has some limitations of imaging infected turbid cornea samples due to increased scattering and does not detect structural alterations in the stroma of infected corneas. TPM is another 3D microscopic imaging technique based on nonlinear two-photon excitation of fluorophores and second harmonic generation (SHG). TPM is less sensitive to increased scattering of turbid cornea sample than CM, and it could apply for corneal diagnosis imaging method. In this study, the aim is to apply TPM imaging technique for diagnosis of corneal infection. This dissertation introduced the use of CM and TPM imaging technique to compare the performance of imaging and diagnosis in various infected cornea sample. First, the performance CM and TPM were compared in the in vivo imaging of mouse corneas in both normal and corneal neovascularization model cases. Second, both imaging technology were also compared in infected rabbit corneas, especially fungal keratitis. Third, I described the applications of moxifloxacin, a FDA-approved fluoroquinolone antibiotic, as a cell labelling agent for TPM imaging to overcome low imaging speed of label-free TPM technique. Finally, the TPM imaging technique was applied for drug delivery study in mouse cornea samples based on two-photon fluorescence. In conclusion, this dissertation presented and suggested the use of TPM imaging method for visualizing various corneal sample. TPM imaging method was applied for showing corneal and vasculature structure in inflamed cornea and infected pathogen in fungal keratitis corneal sample than. TPM provided better contrast with depth than CM due to its lower sensitivity to turbid region in inflamed cornea. Moxifloxacin based TPM imaging technique could overcome the limitations of label-free TPM. Furthermore, TPM technique could be a useful method for studying the pharmacokinetics of fluoroquinolone solutions in the cornea based on their intrinsic fluorescence