Nonlinear optical properties of collagen type-I are investigated in thin tissue sections of pig tendon as a research model using a complete polarimetric second-harmonic generation (P-SHG) microscopy technique called double Stokes-Mueller polarimetry (DSMP). Three complex-valued molecular susceptibility tensor component ratios are extracted. A significant retardance is observed between the chiral susceptibility component and the achiral components, while the achiral components appear to be in phase with each other. The DSMP formalism and microscopy measurements are further used to explain and experimentally validate the conditions required for SHG circular dichroism (SHG-CD) of collagen to occur. The SHG-CD can be observed with the microscope when: (i) the chiral second-order susceptibility tensor component has a non-zero value, (ii) a phase retardance is present between the chiral and achiral components of the second-order susceptibility tensor and (iii) the collagen fibres are tilted out of the image plane. Both positive and negative areas of SHG-CD are observed in microscopy images, which relates to the anti-parallel arrangement of collagen fibres in different fascicles of the tendon. The theoretical formalism and experimental validation of DSMP imaging technique opens new opportunities for ultrastructural characterisation of chiral molecules, in particular collagen, and provides basis for the interpretation of SHG-CD signals. The nonlinear imaging of chiroptical parameters offers new possibilities to further improve the diagnostic sensitivity and/or specificity of nonlinear label-free histopathology.

Second-harmonic generation (SHG) double Stokes-Mueller polarimetric microscopy is applied to study the alteration of collagen ultrastructure in a tissue microarray containing three pathological human breast cancer types with differently overexpressed estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Kleinman symmetry is experimentally validated in breast tissue for 1028 nm laser wavelength and it has been shown that measurements with only linearly polarized incoming and outgoing states can determine molecular nonlinear susceptibility tensor component ratio, average in-plane orientation of collagen fibers and degree of linear polarization of SHG. Increase in the susceptibility ratio for ER, PgR, HER2 positive cases, reveals ultrastructural changes in the collagen fibers while the susceptibility ratio increase and decrease in degree of linear polarization for ER and PgR positive cases indicate alteration of the ultrastructure and increased disorder of the collagen fibers within each focal volume. The study demonstrates a potential use of polarimetric SHG microscopy for collagen characterization and cancer diagnostics. Keely, "Aligned collagen is a prognostic signature for survival in human breast carcinoma," Am. J. Pathol. 178(3),

Femtosecond Laser Two-Photon Polymerization (LTPP) is a fabrication technique based on ultra-localized polymerization reaction initiated by nonlinear absorption of tightly focused light beam. It offers possibility to form three-dimensional (3D) micro-and nanostructures out of photopolymers. The point-by-point photostructuring allows fabrication of objects directly from Computer Aided Design (CAD) models and thereby the geometry of required structure can be changed flexibly. The smallest structural elements, also called voxels (volumetric pixels), of 200 nm lateral dimensions can be achieved with high repeatability. In this article, we present 3D microstructures fabricated out of hybrid zirconium-silicon containing hybrid sol-gel photopolymer ORMOSIL (SZ2080) doped with conventionally used fluorescent dyes: rhodamine 6G (R6G), fluorescein, DCM LC6500, and coumarin 152. The structural quality of the microobjects was investigated by Scanning Electron Microscopy (SEM). Interior of doped 3D micro-and nanostructures has been diagnosed with a custom made scanning fluorescence microscope. Additionally, fluorescing artificial scaffolds, which could be used for cell growth and cell tracking, were manufactured. Finally, the model of Distributed Feedback Dye Laser (DFBL) was successfully fabricated and this demonstrated the possibility to manufacture optically active elements from doped photopolymers.

The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained (H&E) tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor from normal tissue, resulting in 94.2% for both accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.

Polarization-resolvedsecondharmonic generation (P-SHG)