Second harmonic microscopy to quantify renal interstitial fibrosis and arterial remodeling

Non-linear optical microscopy as a novel quantitative and label-free imaging modality to improve the assessment of tissue-engineered cartilage.

In this work, the thermal disruption of type I collagen is investigated using second harmonic generation (SHG) microscopy. We investigated the effects of second harmonic generation images and intensity of rat tail tendon in the temperature range from 25 to 60/spl deg/C. We found a structural transition at 57/spl deg/C where a drop in second harmonic intensity is correlated to a disorganization of the collagen fibers. Our results suggest that SHG microscopy is an effective technique to monitor thermally induced collagen structural changes for biomedical applications.

Non-small cell lung carcinoma (NSCLC) comprises 85% of all lung cancers. Approximately 30-50% of NSCLC recur despite curative resection. There is a major unmet need to identify NSCLC that are likely to recur so that these patients may be treated with more aggressive therapies. Collagen 1 (Col1) fibers are a major structural component of the extracellular matrix (ECM) of tumors. While increased Col1 fibers have been associated with increased metastasis in breast cancer, the role of Col1 in NSCLC and its relationship to recurrence has not been previously examined. We performed a blinded second harmonic generation (SHG) microscopy study on tumor tissue obtained from 5 patients with nonrecurrent NSCLC and 5 patients with recurrent NSCLC. Col1 fibers were detected with SHG microscopy that detects an intrinsic signal derived from the noncentrosymmetric molecular structure of Col1 fibers. SHG microscopy of tumor regions was performed on the H&E sections to quantify Col1 fiber volume and inter fiber distance in the tumors. At least 10 fields of view were analyzed from 1-2 sections obtained from each tumor. We additionally performed Haralick feature analyses to further analyze Col1 fiber patterns. Adjacent sections were immunostained for CD44 and α-SMA to identify cancer associated fibroblasts (CAFs) and understand their role in these tumors. We observed a significant increase of Col1 fiber volume and a significant decrease of inter fiber distance in recurrent NSCLC. Four Haralick features (orientation, contrast, energy and homogeneity) were also significantly higher in the recurrent tumors compared to the non recurrent tumors. We are currently analyzing the CAFs in the tumor sections to understand their role in the significant differences observed in Col1 fibers between recurrent and nonrecurrent tumors. If these preliminary data are confirmed in an expanded sample set, Col1 fiber analysis may provide a companion diagnostic test that can be performed rapidly on H&E tumor sections, using a confocal microscope, to evaluate the likelihood of tumor recurrence. These studies provide new insights into the role of the tumor microenvironment in NSCLC recurrence, and identify new targets for future therapeutic strategies. This work was supported in part by NIH R35CA209960. Citation Format: Samata Kakkad, Peng Huang, Malcolm Brock, Balaji Krishnamachary, Zaver M. Bhujwalla. Collagen 1 fiber content may predict for recurrence in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3691.

Keratoconus is an ophthalmic disease in which the cornea acquires an abnormal conical shape that prevents the correct focusing on the retina, causing visual impairment. The late diagnosis of keratoconus is among the principal causes of corneal transplantation surgery. In several previous studies, a different organization sutural lamellae was observed in keratoconic corneas compared to healthy corneas. This study is intended to investigate this anomaly by means of Second Harmonic Generation (SHG) microscopy, using an optical scheme relevant for clinical applications. In particular, we propose a method based on 3D correlation of SHG image stacks, able to appreciate statistical differences in the orientation distribution of sutural lamellae between healthy and keratoconic corneas. The proposed method offers the potential for diagnosing keratoconus in an early stage.

Second harmonic generation (SHG) microscopy and Raman microscopy were used for qualitative and quantitative analysis of pharmaceutical materials. Prototype instruments and algorithms for sampling strategies and data analyses were developed to achieve pharmaceutical materials analysis with low limits of detection and short measurement timesManufacturing an amorphous solid dispersion (ASD), in which an amorphous active pharmaceutical ingredient (API) within polymer matrix, is an effective approach to improve the solubility and bioavailability of a drug. However, since ASDs are generally metastable materials, they can often transform to produce crystalline API with higher thermodynamic stability. Analytical methods with low limits of detection for crystalline APIs were used to assess the stability of ASDs. With high selectivity to noncentrosymmetric crystals, SHG microscopy was demonstrated as an analytical tool, which exhibited a limit of detection of 10 ppm for ritonavir Form II crystals. SHG microscopy was employed for accelerated stability testing of ASDs, which provided a four-decade dynamic range of crystallinity for kinetic modeling. An established model was validated by investigating nucleation and crystal growth based on SHG images. To achieve in situ accelerated stability testing, controlled environment for in situstability testing (CEiST) was designed and built to provide elevated temperature and humidity, which is compatible with a commercial SHG microscope based on our research prototype. The combination of CEiST and SHG microscopy enabled assessment of individual crystal growth rates by single-particle tracking and nucleation rates for individual fields of view with low Poisson noise. In addition, SHG microscopy coupled with CEiST enabled the study of heterogeneity of crystallization kinetics within pharmaceutical materials.Polymorphism of APIs plays an important role in drug formulation development. Different polymorphs of identical APIs may exhibit different physiochemical properties, e.g., solubility, stability, and bioavailability, due to their crystal structures. Moreover, polymorph transitions may take place during the manufacturing process and storage. Therefore, analytical methods with high speed for polymorph characterization, which can provide real-time feedback for the polymorphic transition, have broad applications in pharmaceutical materials characterization. Raman spectroscopy is able to determine the API polymorphism, but is hampered by the long measurement times. In this study, two analytical methods with high speed were developed to characterize API polymorphs. One is SHGmicroscopy-guided Raman spectroscopy, which achieved the speed of 10 ms/particle for clopidogrel bisulfate. Initial classification of two different polymorphs was based on SHG images, followed acquisition of Raman spectroscopyat the selected positions to determine the API crystal form. Another approach is implementing of dynamic sampling into confocal Raman microscopy to accelerate Raman image acquisition for 6-folds. Instead of raster scanning, dynamic sampling algorithm enabled acquiring Raman spectra at the most informative locations. The reconstructed Raman image of pharmaceutical materials has

Murine induced colon cancer has been used to demonstrate that Second Harmonic Generation (SHG) microscopy images, combined with Two-Photon Excitation Fluorescence (TPEF) and specific quantization scoring methods allow distinguishing early alterations in colon mucosa. TPEF was used only to identified crypts and submucosa regions, whereas the image analysis was used to get quantitative data (Integrated Intensity and Aspect Ratio scoring) of different cancer stages. The submucosa amount of collagen fibers was significant and their orientation suffering proportional changes with the development of the pathological processes. Both after the fourth and eighth weeks after colon cancer induction, integrated intensity and aspect ratio values have shown significant statistical differences compared with control samples. Thus, SHG microscopy has proved to be a useful quantitative tool to highlight early changes of submucosa and the progression of these through the cancer development.

Label-free visualization of cholesteatoma in the mastoid and tympanic membrane using CARS microscopy

By using the second harmonic generation (SHG) microscope, which we have recently developed, two-dimensional images of ferroelectric domain structure were successfully obtained by exploiting the inhomogeneous distribution of the quadratic nonlinear optical constant in specimens. 90° domain structure of BaTiO3 was observed with intensity contrast of the second harmonic wave. A periodically inverted antiparallel domain in LiTaO3 was also observed. This fact indicates that the SHG microscope provides a new method of observing antiparallel ferroelectric domain structure in a nondestructive way, which is especially important for characterizing the quasiphase matched devices for compact blue light lasers.

Optical second harmonic generation (SHG) is a nonlinear optical process which is sensitive to the symmetry of media. SHG microscopy allows for selective probing of a non-centrosymmetric area of sample. This type of nonlinear optical microscope was first used to observe ferroelectric domains and has been applied to various specimens including the biological samples to date. Imaging of the endogenous SHG of biological tissue has been utilized for the selective observation of filament systems in tissues such as collagen, myosin, and microtubules, which exhibit a polar structure. The cellular membrane can be selectively observed by the SHG microscope through membrane staining with amphiphilic polar dye molecules. It has been reported that, by imaging exogenous SHG of the membrane, sensitive detection of membrane damage could be realized using the SHG microscope. Because the staining dye is fluorescent, both SHG and two-photon excited fluorescence (TPF) images can be obtained simultaneously. How the SHG intensity depends on the molecular alignment of the polar dye molecules that reflects the ordering of lipid molecules in the plasma membrane and the necessity of the normalization of the SHG intensity by the TPF intensity is discussed. Furthermore, the assessment of the membrane damage induced by exposing polycation to HeLa cells has been compared with the conventional cytotoxicity and cell viability tests to demonstrate the higher sensitivity of the present SHG-based assay.

Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

Coherent anti-Stokes Raman scattering microscopy.

Keratoconus is an eye disorder that features a reduced stiffness of the cornea and its consequent pathological deformation. Cross-Linking (CXL) treatment has proven useful in hindering the progression of keratoconus, offering a minimally-invasive alternative to corneal surgical transplantation. In this study, the biomechanical characteristics of a human keratoconic cornea were clinically examined in vivo soon before keratoplasty, and the morphological alterations of the collagen scaffold in the same cornea were examined ex vivo by means of Second-Harmonic Generation (SHG) microscopy. A healthy cornea and a CXL-treated keratoconus were compared. In particular, the lamellar organization in the three corneal samples was characterized in different stromal layers by detecting both forward- and backwardscattered SHG signal and then considering the forward/backward (F/B) ratio as parameter. The F/B ratio was used to characterize the morphological organization of collagen lamellae within different stromal layers, finding an increased disorder at the level of Bowman's membrane, opposed to a more regular organization within deeper stromal layers in all the examined samples. The organization of collagen lamellae in CXL-treated keratoconic samples was similar to that one found in healthy corneas, demonstrating that the CXL is able to rearrange the collagen scaffold and partially recover the properties of a healthy condition. The obtained results are in agreement with previous results obtained in studies aimed at monitoring the organization of fibrillar collagen using F/B SHG ratio. In conclusion, the proposed method might be useful for both diagnosing keratoconus as well as for monitoring the effects of the CXL treatment.

Surface crystallization was induced in Ge-doped silica glass samples from a graded-index optical fiber preform by re-heating them at 1100 degrees C for several hours. X-ray diffraction and second-harmonic generation (SHG) microscopy have been utilized to investigate the crystalline phases formed. Experimental results indicate that the predominant crystalline phase is alpha-cristobalite. The cross-sectional distribution of the crystal particles has also been measured with the SHG microscopy and the result is in good agreement with that from traditional bright field light microscopy.

Polarization-resolved second-harmonic generation (P-SHG) microscopy is a technique capable of characterizing nonlinear optical properties of noncentrosymmetric biomaterials by extracting the nonlinear susceptibility tensor components ratio , with z-axis parallel and x-axis perpendicular to the C6 symmetry axis of molecular fiber, such as a myofibril or a collagen fiber. In this paper, we present two P-SHG techniques based on incoming and outgoing circular polarization states for a fast extraction of : A dual-shot configuration where the SHG circular anisotropy generated using incident right- and left-handed circularly-polarized light is measured; and a single-shot configuration for which the SHG circular anisotropy is measured using only one incident circular polarization state. These techniques are used to extract the of myosin fibrils in the body wall muscles of Drosophila melanogaster larva. The results are in good agreement with values obtained from the double Stokes-Mueller polarimetry. The dual- and single-shot circular anisotropy measurements can be used for fast imaging that is independent of the in-plane orientation of the sample. They can be used for imaging of contracting muscles, or for high throughput imaging of large sample areas.

Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.