Modeling nonlinear microscopy near index-mismatched interfaces

Abstract

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.

Kidney Stone Classification Using Multimodal Multiphoton Microscopy

“…The pTHG response shows predominantly single-peak modulation, which indicates that the sample is composed of concentric crystalline subdomains that form vertical interfaces for THG imaging. The pTHG angle distribution is radial, consistent with the presence of concentric index-mismatched vertical interfaces . Such a concentric structure is in line with a pathogenesis process of the calcification that has been described in different organs including kidney, skin, or breast and attributes these micrometer spherical entities made of calcium phosphate apatite to the agglomeration of nanospherules.…”

“…This expression corresponds to a characteristic double-peaked profile as shown in Figure b. In the case of pTHG from an interface parallel to the beam propagation, i.e. , when a linearly polarized excitation beam is focused near a vertical discontinuity of the refractive index, the THG process is significantly altered by field distortions , and cannot be easily described analytically. In high-NA microscopy experiments, the signal is often found to follow a polarization dependence in the form TH G V ( ϕ ) ∝ A + B 0.25em cos 2 ( ϕ − ϕ 0 ) I 3 where A and B are constants.…”

Circular Dichroism Second-Harmonic Generation Imaging of KTiOPO4 Nanocrystal Through Stratified Media

“…In contrast, the FDTD family of methods calculate the electric fields at every point of a 3D grid in successive times by solving discretized Maxwell equations for specified materials. The implementation details and the validity of FDTD strategies in the context of nonlinear microscopy have been demonstrated (Morizet et al, 2021). Figure 4A shows the schematic of an FDTD simulation for SHG microscopy under circular polarized excitation to a vertical interface.…”

“…As a complementary tool with a different principle, the finite-difference time-domain (FDTD) approach has been adopted in many microscopy technique simulations, such as wide-field, confocal, and SHG microscopy (Török et al, 2008;Choi et al, 2007;van der Kolk et al, 2018). More recently, the ubiquitous geometry of a vertical interface between index-mismatched media and the case of polarized THG contrasts are analyzed based on FDTD methods (Morizet et al, 2021). The effects of the refractive index mismatches and the imaging depth on CD-SHG microscopy have not yet been studied.…”

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

“…THG imaging contrast is strongly influenced by wavelength-scale sample heterogeneity, which has been extensively studied 15,[40][41][42][43] . In addition to this dependence on sample microstructure, it has also been shown that biological THG can be enhanced by the presence of strong absorbers such as porphyrin-based pigments.…”