Tissue spectroscopic characterization based on fluorescence, second harmonic generation, and reflected light

Abstract

The diagnosis of many diseases often requires a histological analysis of tissues. Histology analysis compares the microscopic structure of a tissue specimen with an image database containing known physiological and pathological tissue structures. Three new microscopy technologies are developed to complement histology based on novel contrast mechanisms to better visualize and understand tissue structure and function: two-photon spectral resolved imaging, tri-modal imaging, and interferometric second harmonic imaging. First, two-photon spectral resolved microscopy utilizes the 3D localization ability of two-photon excitation to extract spectroscopic information from a femtoliter volume in tissue. The method is capable of the identification of biochemical species in tissues based on their morphological and spectral signatures. This system incorporates two new spectral analysis methods - spectral image guided analysis and multivariate curve resolution. This instrument has been applied to the study of human skin luminescence species and in a photoaging study of a skin equivalent model. Second, tri-modal microscopy combines two-photon fluorescence with second harmonic imaging and reflected light optical coherence microscopy. In this tri-modal system, fluorescence imaging maps fluorophore distribution; second harmonic imaging maps biological crystalline structures such as collagen and microtubules; reflected light optical coherence microscopy maps index of refraction heterogeneity. The ability of this tri- modal microscope has been demonstrated in the imaging of black tetra fish scale and in ex vivo human skin. Third, interferometric second harmonic microscopy has the potential for imaging deeper second harmonic active structures in tissues.

(cont.) This enhancement is based on phase coherent detection allowing the separation of multiple scattered light from the ballistic second harmonic signal. We have implemented interferometric second harmonic microscopy in epi-imaging mode and demonstrated coherent imaging of non-linear optical crystals.