Second Harmonic Generation Imaging Microscopy Research Articles

Enhanced nonlinearities in hybrid structure based on nanoporous membrane and a metallohelicate with promising application in nanophotonics and NLO.

The main purposes of this work are designing new hybrid structures based on alumina nanoporous membranes with specific metallosupramolecular structure as well as studies of their usefulness in nonlinear optics (NLO). The NLO studies of the hybrid material is performed on the basis of two methods: the first by the Maker fringe technique, where the second harmonic generation (SHG) signal is recorded by rotating the sample; and the second by SHG imaging microscopy, where the SHG signal is collected point by point on the sample surface. The enhanced SHG signals were obtained without the use of the corona poling method needed during the experiment on thin films in our previous works and clearly shows the efficiency of hybrid materials based on nanoporous membranes as promising materials in devices developed based on NLO.

Abstract AP19: COLLAGEN ALTERATIONS IN HUMAN OVARIAN CANCER PROBED BY SECOND HARMONIC GENERATION (SHG) IMAGING MICROSCOPY

Abstract PURPOSE: Significant remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer and has been observed in both high grade and low grade tumors as well as ovaries from high risk patients with BRCA mutations. Uniquely quantifying alterations in the tumor microenvironment (TME) could be used as a new diagnostic tool, e.g. to complement histology; provide insight into disease etiology; and assess treatment efficacy through minimally invasive in vivo imaging. Current clinical modalities (CT, MRI, PET) lack the resolution for this purpose. Our efforts have focused on examining collagen alterations across a spectrum of human ovarian tumors, where the changes can serve as quantitative biomarkers. Alterations can be reflected in increased collagen concentration, changes in alignment of collagen molecules within fibrils and/or fibers and/or up-regulation of different collagen isoforms, e.g. Col III. METHODS AND RESULTS: We used the collagen specific/sensitive Second Harmonic Generation (SHG) imaging microscopy to probe collagen architecture over size scales that range from the macromolecular structural properties to fibril/fiber morphology. First, we used SHG polarization analyses to probe collagen macromolecular/supramolecular properties to discriminate ex vivo human tissues (normal stroma, benign tumors, and high grade serous tumors) by determination of: i) collagen alpha helical pitch angle, ii) alignment of collagen molecules within fibrils, and iii) collagen helical chirality via SHG circular dichroism (SHG-CD). The largest differences were between normal stroma and benign tumors, consistent with gene expression data showing Col III is up-regulated in the latter. The different tissues also displayed differing collagen alignment within fibrils and SHG-CD responses, consistent with either Col III incorporation or randomization of Col I alignment within benign and high-grade tumors fibrils. These results collectively indicate the fibril assemblies are distinct in all tissues and likely result from synthesis of new collagen rather than remodeling of existing collagen. Importantly, these techniques do not require exogenous labels, and additionally, provide sub-resolution structural information previously obtained through ultrastructural analysis that cannot be performed on intact tissues. We next implemented a novel form of 3D texture analysis and machine learning to delineate the fibrillar morphology observed in SHG images of normal stroma, high risk stroma, benign tumors, and a spectrum of malignant tumors (high grade serous, low grade serous, and endometrioid). We extracted textural features in the 3D image sets to build statistical models of each class and we achieved clinically significant 83-91% classification accuracies for the six classes. Importantly, the 3D analysis significantly outperformed our prior 2D methods. This classification based on collagen morphology will complement conventional classification based on genetic profiles and can serve as an additional biomarker. CONCLUSIONS: Taken together, the combination of the macro/supramolecular probes and the fiber morphology classification will greatly increase our understanding of the TME evolution in human ovarian cancer. These methods and their findings have clinical translational significance in terms of understanding disease etiology, and also by enhancing prognostic and diagnostic capabilities. Citation Format: Kirby R. Campbell, Rajeev Chaudhary, Julia Handel, Bruce Wen, Vikas Singh, Manish Patankar and Paul J. Campagnola. COLLAGEN ALTERATIONS IN HUMAN OVARIAN CANCER PROBED BY SECOND HARMONIC GENERATION (SHG) IMAGING MICROSCOPY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP19.

Modern methods to differentiate benign thyroid nodules from malignant ones

Objective: The purpose of this study is to compare the capsular structure appearing in follicular adenoma and papillary thyroid carcinoma to differentiate the benign and malignant thyroid nodules. Methods: Second Harmonic Generation (SHG) Microscopy was used to image collagen distribution in the capsules of several types of nodules. The tissue fragments were formalin-fixed, paraffin-embedded but without H&E staining, with 4-5 microns thick sections. From the same sample, two consecutive sections were made. The first was colored with H&E and capsule images were acquired with an optical microscope. The second was not colored with H&E. On the uncolored sample, type I collagen from the capsule was visualized by SHG microscopy. In addition, a parameter was used to study the orientation of collagen fibers, obtained from the Fast Fourier Transform (FFT) analysis of 2D-images. Results: SHG microscopy images were acquired to assess the collagen organization of tumoral capsular thyroid nodules previously diagnosed as benign or malignant by conventional H&E staining. Different degree of collagen fibers orientation was observed from the two kinds of capsules and quantified using FFT analysis. Conclusion: These above described microscopy method can be used to distinguish between benign and malignant thyroid nodules, based on different degree of the capsular collagen fibers orientation.

Quantitative evaluation of atherosclerotic plaques using cross-polarization optical coherence tomography, nonlinear, and atomic force microscopy.

A combination of approaches to the image analysis in cross-polarization optical coherence tomography (CP OCT) and high-resolution imaging by nonlinear microscopy and atomic force microscopy (AFM) at the different stages of atherosclerotic plaque development is studied. This combination allowed us to qualitatively and quantitatively assess the disorganization of collagen in the atherosclerotic arterial tissue (reduction and increase of CP backscatter), at the fiber (change of the geometric distribution of fibers in the second-harmonic generation microscopy images) and fibrillar (violation of packing and different nature of a basket-weave network of fibrils in the AFM images) organization levels. The calculated CP channel-related parameters are shown to have a statistically significant difference between stable and unstable (also called vulnerable) plaques, and hence, CP OCT could be a potentially powerful, minimally invasive method for vulnerable plaques detection.

Quantitative differentiation of normal and scarred tissues using second-harmonic generation microscopy.

The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p < 0.05) than control tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p < 0.005) fiber density (0.72 ± 0.09) compared to controls (0.18 ± 0.03). In the present study, image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as application of steroids or drugs targeting pathways involved in fibrosis. SCANNING 38:684-693, 2016. © 2016 Wiley Periodicals, Inc.

Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy

Purpose: The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states.Methods: To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly.Results: Our results revealed differences in SHG forward–backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella.Conclusions: Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer.

In this perspective, we discuss how the nonlinear optical technique of second-harmonic generation (SHG) microscopy has been used to greatly enhance our understanding of the tumor microenvironment (TME) of breast and ovarian cancer. Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution. This information has not historically been exploited by pathologists but has the potential to enhance diagnostic and prognostic capabilities. We summarize the utility of image processing tools that analyze fiber morphology in SHG images of breast and ovarian cancer in human tissues and animal models. We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues. First we describe the use of polarization-resolved SHG that yields metrics related to macromolecular and supramolecular structures. The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly. These analyses are more general and more broadly applicable than purely morphology-based analyses; however, they are more computationally intensive. Intravital imaging techniques are also emerging that incorporate all of these quantitative analyses. Now, all these techniques can be coupled with rapidly advancing miniaturization of imaging systems to afford their use in clinical situations including enhancing pathology analysis and also in assisting in real-time surgical determination of tumor margins.

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

BackgroundNonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.Methodology/Principal FindingsWe obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.Conclusions/SignificanceNLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

Precision increase with two orthogonal analyzers in polarization-resolved second-harmonic generation microscopy

We analyze the increase in precision of parameters estimation for polarization-resolved second-harmonic generation imaging microscopy when two intensities are measured with two orthogonal analyzers. The analysis is performed for measuring anisotropy parameters and molecule orientation for samples with cylindrical symmetry in the presence of photon noise with Poisson statistics. The improvement in comparison to global intensity measurement (i.e., without analyzer) is discussed.

Hypoxic Tumor Microenvironments Reduce Collagen I Fiber Density

Although the mechanisms through which hypoxia influences several phenotypic characteristics such as angiogenesis, selection for resistance to apoptosis, resistance to radiation and chemotherapy, and increased invasion and metastasis are well characterized, the relationship between tumor hypoxia and components of the extracellular matrix (ECM) is relatively unexplored. The collagen I (Col1) fiber matrix of solid tumors is the major structural part of the ECM. Col1 fiber density can increase tumor initiation, progression, and metastasis, with cancer cell invasion occurring along radially aligned Col1 fibers. Here we have investigated the influence of hypoxia on Col1 fiber density in solid breast and prostate tumor models. Second harmonic generation (SHG) microscopy was used to detect differences in Col1 fiber density and volume between hypoxic and normoxic tumor regions. Hypoxic regions were detected by fluorescence microscopy, using tumors derived from human breast and prostate cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under transcriptional control of the hypoxia response element. In-house fiber analysis software was used to quantitatively analyze Col1 fiber density and volume from the SHG microscopy images. Normoxic tumor regions exhibited a dense mesh of Col1 fibers. In contrast, fewer and structurally altered Col1 fibers were detected in hypoxic EGFP-expressing tumor regions. Microarray gene expression analyses identified increased expression of lysyl oxidase and reduced expression of some matrix metalloproteases in hypoxic compared with normoxic cancer cells. These results suggest that hypoxia mediates Col1 fiber restructuring in tumors, which may impact delivery of macromolecular agents as well as dissemination of cells.

Innovative TCSPC–SHG microscopy imaging to monitor matrix collagen neo-synthetized in bioscaffolds

We propose an innovative invasiveless technique in the field of nonlinear optical imaging to facilitate monitoring of cell/scaffold combinations for tissue repair. By using a near infrared (NIR) femtosecond excitation, we were able to introduce a new index based on decay time response for fluorescence (F) and Second Harmonic Generation (SHG) obtained with Time Correlated Single Photon Counting (TCSPC) microscopy to monitor structural information on the state of the matrix collagen. Some human Mesenchymal Stem Cells (hMSCs) seeded in 3D scaffolds were tested with different culture times (from D7 to D56) to analyze the effect of Tumor Growth Factor beta 1 (TGF-β1) on type-2 collagen expression in the matrix. After 14 days in the presence of TGF-β1, our results showed an increase in the expression of type-2 collagen synthesized by hMSCs, and a change in collagen conformation, as an indication of its ability to be detected as a harmonophore by TCSPC-SHG without the need for an exogenous probe.

Second Harmonic Generation for Visualizing 3-Dimensional Structure of Corneal Collagen Lamellae

Recently, high-energy, short-pulsed femtosecond lasers have been used to generate signals in a process termed second harmonic generation (SHG) specifically from collagen that can be used to obtain high-resolution images of collagen without the need to fix, process, or section tissues. Using SHG imaging microscopy, we have studied the 3-dimensional collagen organization of the cornea. In normal human cornea, 3-dimensional reconstructed images revealed that the organization of collagen lamellae at anterior stroma is highly interwoven and contains prominent transverse “sutural” lamellae that insert into the anterior limiting layer (Bowman layer) and extend deep into the anterior stroma. By comparison, corneal buttons from patients with keratoconus lack sutural lamellae, suggesting that these macroscopic structures may play roles in establishing the structural integrity and biomechanical strength of the anterior cornea and in the pathogenesis of keratoconus. SHG imaging was also used to visualize photodisrupted stromal collagen after IntraLASIK surgery in an animal model and discontinuous collagen lamellae pattern after laser-assisted in situ keratomileusis surgery. Thus, SHG imaging of corneal stromal collagen organization has the potential to provide novel information about corneal physiology and pathobiology.

Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy.

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90 degrees in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 theta distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.