In Vivo Assessment of the Forward to Backward Ratio of Second Harmonic Generation By Collagen As Part of a Non-Invasive, All-Optical Biopsy for Skin Cancer
AbstractThe ratio of forward-to-backward scatter due to Second Harmonic Generation (SHG) in collagen can indicate pathology in skin. SHG, a non-linear optical process where two near-infrared photons of a certain energy are converted to one UV photon of twice the energy at a non-centrosymmetric molecule such as collagen. It has been demonstrated that the forward-to-backward scatter SHG ratio is significantly reduced in cancer biopsy samples. The preferred method uses both forward- and backwardscattered light, requiring a thin biopsy sample. Our goal is to develop and validate an approach to quantify the forward-to-backward SHG ratio in vivo, as part of a non-invasive biopsy for skin cancer. Pulsed 740nm Ti:sapphire light was directed into the epidermis using a laser scanning microscope. The SHG light scattered within the tissue, was di↵usely emitted, and collected by the objective. We then acquired a stack of SHG images using a range of confocal aperture diameters and computed the forward-to-backward SHG ratio using the method by Han and Brown (2010). We used the same process using thin collagen samples to validate our method and correlated the results collected using both methods. Using the results of both methods, we estimated the calibration constant C, the fraction of light initially forward scattered SHG light that arrive at the pinhole aperture. The calibration constant was also estimated using a Monte Carlo simulation. We ran the simulation in a semi-infinite turbid tissue to model an in-vivo measurement and examined C’s dependence on a property of the tissue, the scattering coefficient. We also ran the simulation in a thin biopsy sample to model the collagen samples used experimentally in this project and observed C’s dependence on the scattering coefficient.
In Vivo Imaging, Optical Biopsy, Second Harmonic Generation
Networked Digital Library of Theses and Dissertations