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The development and application of nonlinear optical (NLO) microscopy methods in biomedical research have experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy. 

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research has experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy. 

We study the radiative transfer of a spatially modulated plane wave incident on a half-space composed of a uniformly scattering and absorbing medium. For spatial frequencies that are large compared to the scattering coefficient, we find that first-order scattering governs the leading behavior of the radiance backscattered by the medium. The first-order scattering approximation reveals a specific curve on the backscattered hemisphere where the radiance is concentrated. Along this curve, the radiance assumes a particularly simple expression that is directly proportional to the phase function. These results are inherent to the radiative transfer equation at large spatial frequency and do not have a strong dependence on any particular optical property. Consequently, these results provide the means by which spatial frequency domain imaging technologies can directly measure the phase function of a sample. Numerical simulations using the discrete ordinate method along with the source integration interpolation method validate these theoretical findings.