Characterization of pathological stomach tissue using polarization-sensitive second harmonic generation microscopy.

Alterations in collagen ultrastructure between human gastric adenocarcinoma and normal gastric tissue were investigated using polarization-resolved second harmonic generation (PSHG) microscopy. Cylindrical and trigonal symmetries were assumed to extract quantitative PSHG parameters, ρ, κ and S, from each image pixel. Statistically significant variations in these values were observed for gastric adenocarcinoma, indicating a higher disorder of collagen. Numerical focal volume simulations of crossing fibrils indicate increased S parameter is due to more intersecting collagen fibrils of varying diameters. These parameters were also able to distinguish between different grades of gastric adenocarcinoma indicating that PSHG may be useful for automated cancer diagnosis.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

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.

Investigation into the structure of crystalline maltodextrin particles by second harmonic generation microscopy.

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.

Effect of out of plane orientation on polarization second harmonic generation of single collagen fibrils.

Second harmonic generation (SHG) microscopy has emerged as a powerful technique for visualizing collagen organization within tissues. Amongst the many advantages of SHG is its sensitivity to collagen nanoscale organization, and its presumed sensitivity to the relative out of plane polarity of fibrils. Recent results have shown that circular dichroism SHG (CD-SHG), a technique that has been commonly assumed to reveal the relative out of plane polarity of collagen fibrils, is actually insensitive to changes in fibril polarity. However, results from another research group seem to contradict this conclusion. Both previous results have been based on SHG imaging of collagen fibrils within tissues, therefore, to gain a definitive understanding of the sensitivity of SHG to relative out of plane polarity, the results from individual fibrils are desirable. Here we present polarization resolved SHG microscopy (PSHG) data from individual collagen fibrils oriented out of the image plane by buckling on an elastic substrate. We show through correlation with atomic force microscopy measurements that SHG intensity can be used to estimate the out of plane angle of individual fibrils. We then compare the sensitivity of two PSHG techniques, CD-SHG and polarization-in, polarization-out SHG (PIPO-SHG), to the relative out of plane polarity of individual fibrils. We find that for single fibrils CD-SHG is insensitive to relative out of polarity and we also demonstrate the first direct experimental confirmation that PIPO-SHG reveals the relative out of plane polarity of individual collagen fibrils.

Second harmonic generation microscopy of otoconia.

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.

Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin.

Some osteoblasts embed within bone matrix, change shape, and become dendrite-bearing osteocytes. The circuitry that drives dendrite formation during "osteocytogenesis" is poorly understood. Here we show that deletion of Sp7 in osteoblasts and osteocytes causes defects in osteocyte dendrites. Profiling of Sp7 target genes and binding sites reveals unexpected repurposing of this transcription factor to drive dendrite formation. Osteocrin is a Sp7 target gene that promotes osteocyte dendrite formation and rescues defects in Sp7-deficient mice. Single-cell RNA-sequencing demonstrates defects in osteocyte maturation in the absence of Sp7. Sp7-dependent osteocyte gene networks are associated with human skeletal diseases. Moreover, humans with a SP7R316C mutation show defective osteocyte morphology. Sp7-dependent genes that mark osteocytes are enriched in neurons, highlighting shared features between osteocytic and neuronal connectivity. These findings reveal a role for Sp7 and its target gene Osteocrin in osteocytogenesis, revealing that pathways that control osteocyte development influence human bone diseases.

Buckling and Torsional Instabilities of a Nanoscale Biological Rope Bound to an Elastic Substrate.

Rope-like structures are ubiquitous in Nature. They are supermolecular assemblies of macromolecules responsible for the structural and mechanical integrity of plant and animal tissues. Collagen fibrils with diameters between 50 and 500 nm and their helical supermolecular structure are good examples of such nanoscale biological ropes. Like man-made laid ropes, fibrils are typically loaded in tension, and due to their large aspect ratio, they are, in principle, prone to buckling and torsional instabilities. One way to study buckling of a rigid rod is to attach it to a stretched elastic substrate that is then returned to its original length. In the case of single collagen fibrils, the observed behavior depends on the degree of hydration. By going from buckling in ambient conditions to immersed in a buffer, fibrils go from the well-known sine wave response to a localized behavior reminiscent of the bird-caging of laid ropes. In addition, in ambient conditions, the sine wave response coexists with the formation of loops along the length of the fibrils, as observed for the torsional instability of a twisted filament when tension is decreased. This work provides direct evidence that single collagen fibrils are highly susceptible to axial compression because of their helical supermolecular structure. As a result, mammals that use collagen fibrils as their main load-bearing element in many tissues have evolved mitigating strategies that protect single fibrils from axial compression damage.

Characterization of pathological thyroid tissue using polarization-sensitive second harmonic generation microscopy.

Polarization-sensitive second harmonic generation (SHG) microscopy is an established imaging technique able to provide information related to specific molecular structures including collagen. In this investigation, polarization-sensitive SHG microscopy was used to investigate changes in the collagen ultrastructure between histopathology slides of normal and diseased human thyroid tissues including follicular nodular disease, Grave's disease, follicular variant of papillary thyroid carcinoma, classical papillary thyroid carcinoma, insular or poorly differentiated carcinoma, and anaplastic or undifferentiated carcinoma ex vivo. The second-order nonlinear optical susceptibility tensor component ratios, χ(2)zzz'/χ(2)zxx' and χ(2)xyz'/χ(2)zxx', were obtained, where χ(2)zzz'/χ(2)zxx' is a structural parameter and χ(2)xyz'/χ(2)zxx' is a measure of the chirality of the collagen fibers. Furthermore, the degree of linear polarization (DOLP) of the SHG signal was measured. A statistically significant increase in χ(2)zzz'/χ(2)zxx' values for all the diseased tissues except insular carcinoma and a statistically significant decrease in DOLP for all the diseased tissues were observed compared to normal thyroid. This finding indicates a higher ultrastructural disorder in diseased collagen and provides an innovative approach to discriminate between normal and diseased thyroid tissues that is complementary to standard histopathology.

Live imaging of contracting muscles with wide-field second harmonic generation microscopy using a high power laser.

Wide-field second harmonic generation (SHG) microscopy was developed using a high-power (> 4 W) and high-repetition-rate (MHz range) laser oscillator to achieve fast SHG imaging over a large area (400 µm × 400 µm). The microscope was used for high spatial resolution imaging of contracting muscles in live Drosophila melanogaster larvae. Anisotropic and isotropic bands of striated muscle were distinguished, allowing accurate determination of sarcomere length and SHG intensity from individual sarcomeres. Therefore, wide-field SHG microscopy has applications in basic contractility research and studying arrhythmias, muscular dystrophies and pharmaceutical effects on the muscle contraction dynamics of sarcomeres.

Characterization of Pancreatic Cancer Tissue Using Multiphoton Excitation Fluorescence and Polarization-Sensitive Harmonic Generation Microscopy.

Thin tissue sections of normal and tumorous pancreatic tissues stained with hematoxylin and eosin were investigated using multiphoton excitation fluorescence (MPF), second harmonic generation (SHG), and third harmonic generation (THG) microscopies. The cytoplasm, connective tissue, collagen and extracellular structures are visualized with MPF due to the eosin stain, whereas collagen is imaged with endogenous SHG contrast that does not require staining. Cellular structures, including membranous interfaces and nuclear components, are seen with THG due to the aggregation of hematoxylin dye. Changes in the collagen ultrastructure in pancreatic cancer were investigated by a polarization-sensitive SHG microscopy technique, polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of the SHG signal as a function of the linear polarization orientation of the incident laser radiation. From the PIPO SHG data, the second-order non-linear optical susceptibility ratio, χ(2) zzz '/χ(2) zxx ', was obtained that serves as a structural parameter for characterizing the tissue. Furthermore, by assuming C6 symmetry, an additional second-order non-linear optical susceptibility ratio, χ(2) xyz '/χ(2) zxx ', was obtained, which is a measure of the chirality of the collagen fibers. Statistically-significant differences in the χ(2) zzz '/χ(2) zxx ' values were found between tumor and normal pancreatic tissues in periductal, lobular, and parenchymal regions, whereas statistically-significant differences in the full width at half maximum (FWHM) of χ(2) xyz '/χ(2) zxx ' occurrence histograms were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Additionally, the PIPO SHG data were used to determine the degree of linear polarization (DOLP) of the SHG signal, which indicates the relative linear depolarization of the signal. Statistically-significant differences in DOLP values were found between tumor and normal pancreatic tissues in periductal and parenchymal regions. Hence, the differences observed in the χ(2) zzz '/χ(2) zxx ' values, the FWHM of χ(2) xyz '/χ(2) zxx ' values and the DOLP values could potentially be used to aid pathologists in diagnosing pancreatic cancer.

Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X-Linked Hypophosphatemia.

Osteocytes remodel their surrounding perilacunar matrix and canalicular network to maintain skeletal homeostasis. Perilacunar/canalicular remodeling is also thought to play a role in determining bone quality. X-linked hypophosphatemia (XLH) is characterized by elevated serum fibroblast growth factor 23 (FGF23) levels, resulting in hypophosphatemia and decreased production of 1,25 dihydroxyvitamin D (1,25D). In addition to rickets and osteomalacia, long bones from mice with XLH (Hyp) have impaired whole-bone biomechanical integrity accompanied by increased osteocyte apoptosis. To address whether perilacunar/canalicular remodeling is altered in Hyp mice, histomorphometric analyses of tibia and 3D intravital microscopic analyses of calvaria were performed. These studies demonstrate that Hyp mice have larger osteocyte lacunae in both the tibia and calvaria, accompanied by enhanced osteocyte mRNA and protein expression of matrix metalloproteinase 13 (MMP13) and genes classically used by osteoclasts to resorb bone, such as cathepsin K (CTSK). Hyp mice also exhibit impaired canalicular organization, with a decrease in number and branching of canaliculi extending from tibial and calvarial lacunae. To determine whether improving mineral ion and hormone homeostasis attenuates the lacunocanalicular phenotype, Hyp mice were treated with 1,25D or FGF23 blocking antibody (FGF23Ab). Both therapies were shown to decrease osteocyte lacunar size and to improve canalicular organization in tibia and calvaria. 1,25D treatment of Hyp mice normalizes osteocyte expression of MMP13 and classic osteoclast markers, while FGF23Ab decreases expression of MMP13 and selected osteoclast markers. Taken together, these studies point to regulation of perilacunar/canalicular remodeling by physiologic stimuli including hypophosphatemia and 1,25D. © 2017 American Society for Bone and Mineral Research.

Examination of Drosophila eye development with third harmonic generation microscopy.

Third harmonic generation (THG) microscopy can exploit endogenous harmonophores such as pigment macromolecules for enhanced image contrast, and therefore can be used without exogenous contrast agents. Previous studies have established that carotenoid compounds are ideal harmonophores for THG microscopy; we therefore sought to determine whether THG from endogenous carotenoid-derived compounds, such as retinal in photoreceptor cells, could serve as a new label-free method for developmental studies. Here we study the development of the pupal eye in Drosophila melanogaster and determine the localization of rhodopsin using THG microscopy technique. Additionally, by altering the chromophore or the opsin protein we were able to detect changes in both the retinal distribution morphology and in THG intensity age-dependent profiles. These results demonstrate that THG microscopy can be used to detect altered photoreceptor development and may be useful in clinically relevant conditions associated with photoreceptor degeneration.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Changes of collagen ultrastructure in breast cancer tissue determined by second-harmonic generation double Stokes-Mueller polarimetric microscopy.

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.

Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration.

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'SHG ) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'SHG values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices. The maximum SHG intensity occurs at the highest moisture and amylopectin content. These results support the hypothesis that SHG is caused by ordered hydrogen and hydroxyl bond networks which increase with hydration of starch granules.

Ultrastructural features of collagen in thyroid carcinoma tissue observed by polarization second harmonic generation microscopy.

Changes in collagen ultrastructure between malignant and normal human thyroid tissue were investigated ex vivo using polarization second harmonic generation (SHG) microscopy. The second-order nonlinear optical susceptibility tensor component ratio and the degree of linear polarization (DOLP) of the SHG signal were measured. The ratio values are related to the collagen ultrastructure, while DOLP indicates the relative amount of coherent signal and incoherent scattering of SHG. Increase in ratio values and decrease in DOLP were observed for tumor tissue compared to normal thyroid, indicating higher ultrastructural disorder in tumor collagen.

Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy.

Nonlinear optical microscopy has become a powerful tool for high-resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo-damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure-associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure-dependent switch between harmonic (OAP-intact) and fluorescence (OAP-disrupted) generation enabled real-time multi-modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre-formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging.

Second Harmonic Generation Mediated by Aligned Water in Starch Granules.

The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.

Crystal lattice determination of ZnSe nanowires with polarization-dependent second harmonic generation microscopy.

We demonstrate a noninvasive optical microscopy technique based on polarization-dependent second harmonic generation for determining the crystal lattice structure and microscopic heterogeneities within individual nanostructures. Differentiation between periodically twinned and wurtzite ZnSe nanowires (NWs) was demonstrated, and measurement of the cubic lattice rotation orientation around the NW axis was determined within 1° accuracy. Zinc blende NWs were differentiated from wurtzite. The technique can be used for quality inspection and optimization of growth conditions for nanostructures.