Quantitative analysis of multiphoton excitation autofluorescence and second harmonic generation imaging for medical diagnosis.

In recent years, two-photon excitation fluorescence and second harmonic generation microscopy has become an important tool in biomedical research. The ability of two-photon microscopy to achieve optical sectioning with minimal invasiveness is particularly advantageous for biomedical diagnosis. Advances in the miniaturization of the imaging system have increased its clinical potential, together with the development of quantitative technique for the analysis of data acquired using these imaging modalities. We present a review of the quantitative analysis techniques that have been used successfully with two-photon excitation fluorescence and SHG imaging. Specifically, quantification techniques using ratiometric, morphological, and structural differences to analyze two-photon images will be discussed, and their effectiveness at evaluating dermal and corneal pathologies and cancerous tumor growth will be described.