Optical biopsy has been shown to discriminate between normal and diseased tissue with high sensitivity and specificity. Fiber-optic probe-based spectroscopy systems do not provide the necessary spatial information to guide therapy effectively, ultimately requiring a transition from probe-based spectroscopy to spectral imaging. The effect of such a transition on fluorescence and diffuse reflectance line shape is investigated. Inherent differences in spectral line shape between spectroscopy and imaging are characterized and many of these differences may be attributed to a shift in illumination–collection geometry between the two systems. Sensitivity of the line-shape disparity is characterized with respect to changes in sample absorption and scattering as well as to changes in various parameters of the fiber-optic probe design (e.g., fiber diameter, beam steering). Differences in spectral line shape are described in terms of the relative relationship between the light diffusion within the tissue and the distribution of source–detector separation distances for the probe-based and imaging illumination–collection geometries. Monte Carlo simulation is used to determine fiber configurations that minimize the line-shape disparity between the two systems. In conclusion, we predict that fiber-optic probe designs that mimic a spectral imaging geometry and spectral imaging systems designed to emulate a probe-based geometry will be difficult to implement, pointing toward a posteriori correction for illumination–collection geometry to reconcile imaging and probe-based spectral line shapes or independent evaluation of tissue discrimination accuracy for probe-based and spectral imaging systems.
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