Nonlinear microscopy is widely used to characterize thick, optically
heterogeneous biological samples. While quantitative image analysis
requires accurately describing the contrast mechanisms at play, the
majority of established numerical models neglect the influence of
field distortion caused by sample heterogeneity near focus. In this
work, we show experimentally and numerically that finite-difference
time-domain (FDTD) methods are applicable to model focused fields
interactions in the presence of heterogeneities, typical of nonlinear
microscopy. We analyze the ubiquitous geometry of a vertical interface
between index-mismatched media (water, glass, and lipids) and consider
the cases of two-photon-excited fluorescence (2PEF), third-harmonic
generation (THG) and polarized THG contrasts. We show that FDTD
simulations can accurately reproduce experimental images obtained on
model samples and in live adult zebrafish, in contrast with previous
models neglecting field distortions caused by index mismatch at the
micrometer scale. Accounting for these effects appears to be
particularly critical when interpreting coherent and
polarization-resolved microscopy data.
The paper deals with the specular optical properties of thin dielectric or metallic multilayer coatings. Recurrent formulas are given for reflectance, transmittance, and their derivatives with respect to thicknesses, indices, incidence, and wavelength. Because of its simplicity and flexibility, the proposed method is particularly well fitted for use in a digital computer and for optimization programs.
Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2$$'$$
′
-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarization-resolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries.
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