Polarized light microscopy provides a signal specific to the molecular order. Nonlinear interaction of the light with specimen provides contrast that is specific to the structural or molecular property. All nonlinear methods are attractive for imaging tissues with high signal to noise ratio, because of their intrinsic sectioning property, which arises because the high excitation amplitude required to produce nonlinear interaction is present only at the focus. The present paper by Muhnoz, Rigneault, and Brasselet combines these two advantages to propose and demonstrate a polarization-resolved four-wave mixing microscopy. Four-wave mixing (FWM) is an important addition to the toolbox of well-explored nonlinear imaging methods of two-photon fluorescence (TPF), second harmonic generation (SHG), third harmonic generation (THG), and coherent anti-stokes Raman scattering (CARS). TPF provides contrast by exciting fluorescence, SHG provides contrast for noncentrosymmetric structures, THG provides contrast for interfaces, and CARS provides contrast for vibrational states of molecules. FWM is a non-resonant counterpart of CARS and provides topological contrast that depends on variation of the refractive index. FWM is intriguing, because it does not require noncentrosymmetry or interface or presence of specific molecular vibration. Thus, FWM can provide depth-resolved topological information.
The authors have enhanced FWM's utility by investigating the nature of information it captures. As discussed theoretically in the paper, with polarization-resolved FWM, one can probe even-order terms of the angular distribution of molecules. With rat tendon (a specimen rich in collagen), the authors demonstrate the measurement process. The intriguing approach of measuring depth-resolved structural information presented in this paper should be useful for specimens where SHG and THG do not provide adequate structural contrast.
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