The advancement of non-invasive quantitative optical diagnosis techniques such as polarization-sensitive second harmonic generation microscopy (PSHG) for diseases such as cancer presents opportunities for improving disease understanding and survival rates. Here, novel and developing techniques in PSHG microscopy applied for the differentiation of cancerous or diseased tissues are presented, including circular dichroism, modulation of laser linear polarization, detection of outgoing linear laser polarization, and double-Stokes Mueller. Typically, initial cancer diagnosis is performed by visual inspection of stained biopsy or surgical resection tissue sections under bright-field microscopy, however, early diagnosis is challenging due to variability in morphological interpretation of the tissues, and because cancer initiation regions can be small and easy to miss. Therefore, pathologists could benefit in identifying cancer on biopsy or surgical resection sections by using unbiased quantitative automated technologies with high spatial resolution and improved disease specificity that can check the entire slide pixel-by-pixel. Second harmonic generation microscopy offers the opportunity to measure ultrastructural alterations in collagenous scaffolds of organ tissues virtually background free on submicron-sized tissue regions. The approach is particularly interesting for cancer diagnosis applications, because during cancer initiation and progression, the collagen in the affected tissue extracellular matrix is often deregulated and becomes disorganized. This mini-review contains a thorough summary of PSHG techniques that have interrogated diseased tissues, and discusses their technical variations and successes in disease discrimination.
Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.
The origin of second harmonic generation (SHG) signal in otoconia was investigated. SHG signal intensity from otoconia was compared to pure calcite crystals, given calcite is the primary component of otoconia and is known to emit surface SHG. The SHG intensity from calcite was found to be ∼41× weaker than the SHG intensity from otoconia signifying that the SHG signal from otoconia is likely generated from the organic matrix. Furthermore, the SHG intensity from otoconia increased when treated with a chelating agent known to dissolve calcite which confirms that calcite is not the source of SHG. Additionally, polarization-resolved SHG microscopy imaging revealed that the arrangement of the SHG emitters is radial and can form highly ordered domains.
Crystalline maltodextrin particles (CMPs) were investigated using polarization-sensitive second harmonic generation (PSHG) microscopy to determine changes in their crystalline organization due to crystal type (A- and B-type) and hydration for application as starch model systems. Optimization of their synthesis resulted in intense SHG emission, exceeding maize starch granules. PSHG data showed that CMPs have a radial macrostructure with respect to their nucleation regions, fitted ρ values of 2-6, and some similar hydration variations, mimicking starch granules and validating that CMPs may be used as a model system for improved understanding of the SHG properties and applications of starch granules.
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