Multiphoton microscopy for imaging infectious keratitis: demonstration of the pattern of microbial spread in an experimental model

The purpose of this study is to assess the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model. Porcine eyes are to be obtained from slaughter house and processed and placed in organ culture system. Fungal infections by common pathogens of infectious keratitis are to be induced in porcine cornea buttons. Multiphoton fluorescence and SHG microscopy will be used for imaging and for monitoring the progression and extension of tissue destruction and possibly the pattern of pathogen spreading. We found that SHG imaging is useful in identifying alterations to collagen architecture while autofluorescence microscopy can be used to visualize the fungi and cells within the stroma. In summary, multiphoton fluorescence and second harmonic generation microscopy can non-invasively demonstrate and monitor tissue destruction associated with infectious keratitis. The pattern of pathogen spreading and its correlation with the tissue architecture can also be shown, which can be useful for future studies of the tissue-microbial interactions for infectious keratitis.