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The microstructural analysis of tissues plays a crucial role in the early detection of abnormal tissue morphology. Polarization microscopy, an optical tool for studying the anisotropic properties of biomolecules, can distinguish normal and malignant tissue features even in the absence of exogenous labelling. To facilitate the quantitative analysis, we developed a polarization-sensitive label-free imaging system based on the Stokes-Mueller calculus. Polarization images of ductal carcinoma tissue samples were obtained using various input polarization states and Stokes-Mueller images were reconstructed using Matlab software. Further, polarization properties, such as degree of linear and circular polarization and anisotropy, were reconstructed from the Stokes images. The Mueller matrix obtained was decomposed using the Lu-Chipman decomposition method to acquire the individual polarization properties of the sample, such as depolarization, diattenuation and retardance. By using the statistical parameters obtained from the polarization images, a support vector machine (SVM) algorithm was trained to facilitate the tissue classification associated with its pathological condition.

This paper addresses the application of multimodal nonlinear optical (MNLO) microscopy to clinical research within the context of label-free non-invasive molecular imaging. Here, a compact MNLO microscope based on a laser scanning microscope, a femtosecond laser, a time-correlated single-photon counting system, and a photonic crystal fiber are introduced for biomedical applications. By integrating two-photon fluorescence, two-photon fluorescence lifetime imaging, second-harmonic generation, and coherent anti-Stokes Raman scattering microscopy, the proposed scheme provides profound insights into the physicochemical properties related to 3D molecular orientation distribution, inter- and intra-molecular interactions, and disease progression in biological systems and organs. The high peak power and the low average intensity of near-infrared laser pulses allow for deep-penetration imaging without compromising sample vitality. Linking nonlinear optical phenomena with time/spectral/polarization-resolved imaging also makes it possible to obtain multidimensional information to address complex biomedical questions.

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