Second Harmonic Generation Microscopy Research Articles

Second harmonic generation microscopy of electromechanical reshaping on corneal collagen

Refractive errors remain a global health concern, as a large proportion of the world's population is myopic. Current ablative approaches are costly, not without risks, and not all patients are candidates for these procedures. Electromechanical reshaping (EMR) has been explored as a viable cost-effective modality to directly shape tissues, including cartilage. In this study, stromal collagen structure and fibril orientation was examined before and after EMR with second-harmonic generation microscopy (SHG), a nonlinear multiphoton imaging method that has previously been used to study native corneal collagen with high spatial resolution. EMR, using a milled metal contact lens and potentiostat, was performed on the corneas of five extracted rabbit globes. SHG was performed using a confocal microscopy system and all images underwent collagen fibril orientation analysis. The collagen SHG signal in controls is uniform and is similarly seen in samples treated with pulsed potential, while continuous EMR specimens have reduced, nonhomogeneous signal. Collagen fibril orientation in native tissue demonstrates a broad distribution with suggestion of another peak evolving, while with EMR treated eyes a bimodal characteristic becomes readily evident. Pulsed EMR may be a means to correct refractive errors, as when comparing its SHG signal to negative control, preservation of collagen structures with little to no damage is observed. From collagen fiber orientation analysis, it can be inferred that simple DC application alters the structure of collagen. Future studies will involve histological assessment of these layers and multi-modal imaging analysis of dosimetry.

Contrast-enhanced phase-resolved second harmonic generation microscopy.

The characterization of inverted structures (crystallographic, ferroelectric, or magnetic domains) is crucial in the development and application of novel multi-state devices. However, determining these inverted structures needs a sensitive probe capable of revealing their phase correlation. Here a contrast-enhanced phase-resolved second harmonic generation (SHG) microscopy is presented, which utilizes a phase-tunable Soleil-Babinet compensator and the interference between the SHG fields from the inverted structures and a homogeneous reference. By this means, such inverted structures are correlated through the π-phase difference of SHG, and the phase difference is ultimately converted into the intensity contrast. As a demonstration, we have applied this microscopy in two scenarios to determine the inverted crystallographic domains in two-dimensional van der Waals material MoS2. Our method is particularly suitable for applying in vacuum and cryogenic environments while providing optical diffraction-limited resolution and arbitrarily adjustable contrast. Without loss of generality, this contrast-enhanced phase-resolved SHG microscopy can also be used to resolve other non-centrosymmetric inverted structures, e.g. ferroelectric, magnetic, or multiferroic phases.

A comparative study of CARE 2D and N2V 2D for tissue-specific denoising in second harmonic generation imaging.

This study explored the application of deep learning in second harmonic generation (SHG) microscopy, a rapidly growing area. This study focuses on the impact of glycerol concentration on image noise in SHG microscopy and compares two image restoration techniques: Noise-to-Void 2D (N2V 2D, no reference image restoration) and content-aware image restoration (CARE 2D, full reference image restoration). We demonstrated that N2V 2D effectively restored the images affected by high glycerol concentrations. To reduce sample exposure and damage, this study further addresses low-power SHG imaging by reducing the laser power by 70% using deep learning techniques. CARE 2D excels in preserving detailed structures, whereas N2V 2D maintains natural muscle structure. This study highlights the strengths and limitations of these models in specific SHG microscopy applications, offering valuable insights and potential advancements in the field .

Abstract 5149: Extracellular matrix remodeling in metastatic mismatch repair deficient endometrial cancer:Implications for immune checkpoint inhibitor response prediction

Abstract Mismatch repair deficient (MMRd) status is a predictive biomarker for Immune checkpoint inhibitors (ICI) in endometrial cancer (EC), however, half of these patients (pts) do not respond. Most studies exploring biomarkers of response to ICI have focused on tumor or immune cell factors. We aimed to describe the extracellular matrix components of the tumor microenvironment (TME) in ICI-Responder (R) versus Non-Responder (NR) MMRd EC pts to identify new predictive biomarkers of response. Clinical data and outcomes of metastatic MMRd EC pts, treated with ICI (2016-2021), were retrospectively collected. Pts were classified as Rs (CR, PR, or SD ≥12 months) or NRs (PD or SD <12 months). Pre-ICI FFPE tumor samples were subjected to Biognosys UltraDeep TrueDiscovery™ Mass Spectrometry (MS) for identification of differentially regulated protein expression between R and NR. Criteria for protein candidate selection were p-value < 0.01 and average absolute log2 fold change (AVG Log ratio) > 0.58. Collagen was visualized on whole slides using Masson’s trichrome and quantified by Qupath. Collagen density was expressed as % positive surface in the intratumoral area. Second-harmonic generation (SHG) microscopy plus polarimetry (p-SHG) was used to estimate collagen content and describe the spatial orientation of fibrillar collagen in the intratumoral area. intraepithelial (ie) and stromal (s) CD3+ and CD8+ cells were quantified by Qupath. Non-parametric t-tests and Spearman’s rank coefficient (r) for linear correlation were used. Twenty-five tumor samples were included in the analysis: 17 from Rs and 8 from NRs. MS identified collagen type V alpha-2 (AVR Log ratio 2.9, p 0.008) and type XXII alpha-1 chains (AVR Log ratio 1.14, p 0.006) as significantly upregulated in NRs. Using spatial imaging, collagen density in the intratumoral area was significantly higher in NRs (median: 14.2% vs 4.6%, p 0.01). Intratumoral collagen density was positively correlated with collagen type V expression (r 0,54, p 0.016), and negatively correlated with ie and s CD3+ cell (r -0.39, p 0.054; r -0.4, p 0.048, respectively) and ie and s CD8+ cell (r -0.39, p 0.053; and r -0.29, p 0.166) infiltration. SHG confirmed that fibrillar collagen content in the tumor area was significantly greater in NRs (p 0.02). p-SHG revealed that NR tumors tended to show a highly disorganized collagen matrix. In contrast, a linear collagen pattern was associated with response to ICI: 89% of tumors displaying this pattern were Rs. Both MS and two different spatial approaches consistently demonstrated that increased collagen expression or density may serve as a potential predictive biomarker of resistance to ICI in MMRd metastatic EC, contributing to create an immunosuppressive TME. The spatial orientation pattern of collagen fibers may also be crucial in shaping immune TME and influencing the response to ICI. Citation Format: Juan Francisco Grau, Carole Aimé, Martin Mehnert, Elisa Yaniz-Galende, Catherine Genestie, Audrey Le Formal, Elodie Edmond, Antoine Amaury Lachaud, Patricia Pautier, Judith Michels, Sébastien Gouy, Amandine Maulard, Emeline Colomba-Blameble, Felix Blanc-Durand, Marie-Claire Schanne-Klein, Alexandra Leary. Extracellular matrix remodeling in metastatic mismatch repair deficient endometrial cancer:Implications for immune checkpoint inhibitor response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5149.

Polarization-resolved super-resolution second-harmonic generation imaging based on multifocal structured illumination microscopy.

Polarization-resolved second-harmonic generation (PSHG) microscopy is widely used in investigating the structural and morphological alterations of collagen. However, the resolution of second-harmonic generation (SHG) imaging remains constrained by optical diffraction, resulting in the polarization extraction of collagen characteristics from the average properties of collagen fibers. In this study, multifocal structured illumination microscopy (MSIM) was combined with PSHG to achieve polarization-resolved super-resolution imaging of second-harmonic generation signals. For the first time to our knowledge, periodic structures with an average pitch of 277 nm were observed in mouse tail tendons using optical microscopy, and the orientation angle of fibrils within each period was found to exhibit an alternating arrangement along the axis in a regular pattern.

Label-Free In Vivo Monitoring of Lipid Droplets for Real-Time Assessment of Adipose Activator-Induced Tumor Suppression.

To enhance our comprehension of the fundamental mechanisms driving tumor metabolism and metastasis, it is essential to dynamically monitor intratumoral lipid droplet (LD) and collagen processes in vivo. Traditional LD analysis in tumors predominantly relies on observations of in vitro cells or tissue slices, which unfortunately hinder real-time insights into the dynamic behavior of LDs during in vivo tumor progression. In this study, we developed a dual-modality imaging technique that combines coherent anti-Stokes Raman scattering (CARS) and second-harmonic generation (SHG) microscopy for in vivo monitoring of tumor LDs and collagen alterations, assisted by a murine breast cancer 4T1 cell-based dorsal skinfold window. Specifically, we accomplished real-time observations and quantitative analysis of the LD size, density, and collagen alignment within living tumors through CARS/SHG imaging. Additionally, our findings demonstrate that real-time LD monitoring provides a valuable means of assessing the efficacy of anticancer drugs in vivo. We evaluated the impact of adipose activators on lipid metabolism, oxidative stress, and tumor suppression by monitoring changes in LD size and density. Overall, this study highlights the potential of dual-modality CARS/SHG microscopy as a sensitive and flexible tool for antitumor therapeutic strategies.

Modulation of Surface Plasmon due to the Inhomogeneity of a One-Dimensional Linear Array of Gold Nanoparticles Observed by Optical Second-Harmonic Generation Microscopy

Modulation of Surface Plasmon due to the Inhomogeneity of a One-Dimensional Linear Array of Gold Nanoparticles Observed by Optical Second-Harmonic Generation Microscopy

Second-harmonic generation microscopy with synthetic aperture and computational adaptive optics

Second-harmonic generation (SHG) microscopy is a powerful label-free imaging tool widely used to visualize collagen and muscle in biological tissues. However, traditional laser-scanning SHG microscopy requiring voxel scanning is time-intensive. Wide-field SHG microscopy was designed to bypass this restriction, but its application to deep tissue imaging is limited due to vulnerability to scattering and sample-induced aberrations. We introduce synthetic aperture SHG (SA-SHG) microscopy to attenuate the effect of multiple scattering noises. Our SA-SHG method coherently integrates amplitude and phase maps of wide-field SHG fields taken for different illumination angles, thereby enhancing the signal-to-noise ratio. We also develop computational adaptive optics SHG (CAO-SHG) microscopy to computationally correct the sample-induced aberrations. Our algorithm optimizes SHG fields’ aperture synthesis to identify aberration maps, enabling the restoration of diffraction-limited imaging. We successfully apply this approach to real biological samples, demonstrating its potential for high-resolution imaging in complex biological environments.

Functional Assessment of Human Articular Cartilage Using Second Harmonic Generation (SHG) Imaging: A Feasibility Study.

Many arthroscopic tools developed for knee joint assessment are contact-based, which is challenging for in vivo application in narrow joint spaces. Second harmonic generation (SHG) laser imaging is a non-invasive and non-contact method, thus presenting an attractive alternative. However, the association between SHG-based measures and cartilage quality has not been established systematically. Here, we investigated the feasibility of using image-based measures derived from SHG microscopy for objective evaluation of cartilage quality as assessed by mechanical testing. Human tibial plateaus harvested from nine patients were used. Cartilage mechanical properties were determined using indentation stiffness (Einst) and streaming potential-based quantitative parameters (QP). The correspondence of the cartilage electromechanical properties (Einst and QP) and the image-based measures derived from SHG imaging, tissue thickness and cell viability were evaluated using correlation and logistic regression analyses. The SHG-related parameters included the newly developed volumetric fraction of organised collagenous network (Φcol) and the coefficient of variation of the SHG intensity (CVSHG). We found that Φcol correlated strongly with Einst and QP (ρ = 0.97 and - 0.89, respectively). CVSHG also correlated, albeit weakly, with QP and Einst, (|ρ| = 0.52-0.58). Einst and Φcol were the most sensitive predictors of cartilage quality whereas CVSHG only showed moderate sensitivity. Cell viability and tissue thickness, often used as measures of cartilage health, predicted the cartilage quality poorly. We present a simple, objective, yet effective image-based approach for assessment of cartilage quality. Φcol correlated strongly with electromechanical properties of cartilage and could fuel the continuous development of SHG-based arthroscopy.

In-Plane Ferrielectric Order in van der Waals β'-In2Se3.

van der Waals ferroic materials exhibit rich potential for implementing future generation functional devices. Among these, layered β'-In2Se3 has fascinated researchers with its complex superlattice and domain structures. As opposed to ferroelectric α-In2Se3, the understanding of β'-In2Se3 ferroic properties remains unclear because ferroelectric, antiferroelectric, and ferroelastic characteristics have been separately reported in this material. To develop useful applications, it is necessary to understand the microscopic structural properties and their correlation with macroscopic device characteristics. Herein, using scanning transmission electron microscopy (STEM), we observed that the arrangement of dipoles deviates from the ideal double antiparallel antiferroelectric character due to competition between antiferroelectric and ferroelectric structural ordering. By virtue of second-harmonic generation, four-dimensional STEM, and in-plane piezoresponse force microscopy, the long-range inversion-breaking symmetry, uncompensated local polarization, and net polarization domains are unambiguously verified, revealing β'-In2Se3 as an in-plane ferrielectric layered material. Additionally, our device study reveals analogous resistive switching behaviors of different types owing to polarization switching, defect migration, and defect-induced charge trapping/detrapping processes.

Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy

Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0–F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.

A Universal Strategy for Synthesis of 2D Ternary Transition Metal Phosphorous Chalcogenides.

The 2D ternary transition metal phosphorous chalcogenides (TMPCs) have attracted extensive research interest due to their widely tunable band gap, rich electronic properties, inherent magnetic and ferroelectric properties. However, the synthesis of TMPCs via chemical vapor deposition (CVD) is still challenging since it is difficult to control reactions among multi-precursors. Here, a subtractive element growth mechanism is proposed to controllably synthesize the TMPCs. Based on the growth mechanism, the TMPCs including FePS3 , FePSe3 , MnPS3 , MnPSe3 , CdPS3 , CdPSe3 , In2 P3 S9 , and SnPS3 are achieved successfully and further confirmed by Raman, second-harmonic generation (SHG), and scanning transmission electron microscopy (STEM). The typical TMPCs-SnPS3 shows a strong SHG signal at 1064nm, with an effective nonlinear susceptibility χ(2) of 8.41×10-11 mV-1 , which is about 8 times of that in MoS2 . And the photodetector based on CdPSe3 exhibits superior detection performances with responsivity of 582mAW-1 , high detectivity of 3.19×1011 Jones, and fast rise time of 611µs, which is better than most previously reported TMPCs-based photodetectors. These results demonstrate the high quality of TMPCs and promote the exploration of the optical properties of 2D TMPCs for their applications in optoelectronics.

Collagen fiber centerline tracking in fibrotic tissue via deep neural networks with variational autoencoder-based synthetic training data generation.

The role of fibrillar collagen in the tissue microenvironment is critical in disease contexts ranging from cancers to chronic inflammations, as evidenced by many studies. Quantifying fibrillar collagen organization has become a powerful approach for characterizing the topology of collagen fibers and studying the role of collagen fibers in disease progression. We present a deep learning-based pipeline to quantify collagen fibers' topological properties in microscopy-based collagen images from pathological tissue samples. Our method leverages deep neural networks to extract collagen fiber centerlines and deep generative models to create synthetic training data, addressing the current shortage of large-scale annotations. As a part of this effort, we have created and annotated a collagen fiber centerline dataset, with the hope of facilitating further research in this field. Quantitative measurements such as fiber orientation, alignment, density, and length can be derived based on the centerline extraction results. Our pipeline comprises three stages. Initially, a variational autoencoder is trained to generate synthetic centerlines possessing controllable topological properties. Subsequently, a conditional generative adversarial network synthesizes realistic collagen fiber images from the synthetic centerlines, yielding a synthetic training set of image-centerline pairs. Finally, we train a collagen fiber centerline extraction network using both the original and synthetic data. Evaluation using collagen fiber images from pancreas, liver, and breast cancer samples collected via second-harmonic generation microscopy demonstrates our pipeline's superiority over several popular fiber centerline extraction tools. Incorporating synthetic data into training further enhances the network's generalizability. Our code is available at https://github.com/uw-loci/collagen-fiber-metrics.

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.

TMIC-03. TARGETING THE COLLAGEN RECEPTOR DDR1 PROMOTES ANTI-GLIOMA IMMUNITY BY REMODELING COLLAGEN FIBER ARCHITECTURE

Abstract Glioblastoma (GBM), characterized by its aggressive nature and profound immunosuppression, poses a significant therapeutic challenge with limited treatment options. We previously demonstrated that targeting Col1α1 eradicates oncostreams and improves survival in murine models. However, the underlying mechanisms by which collagen reshapes the glioma tumor microenvironment (TME) remain unknown. Through RNA-seq analysis we identified that DDR1, a collagen receptor, is overexpressed in genetically engineered glioma mouse models (GEMMs) including NPA (NRAS/shp53/shATRX), and NPD (NRAS/shp53/PDGFβ), compared to healthy brain tissue. Pharmacological inhibition of DDR1 enhanced radiosensitivity, disrupted oncostreams in vitro, and ex vivo, imaged with time-lapse confocal microscopy. GEMMs of DDR1 knockdown (NRAS/shp53/shATRX/shDDR1) using the Sleeping Beauty transposase system significantly increased median survival by over 50%. Gene Ontology analysis of differentially expressed genes between wild-type and DDR1 knockdown gliomas revealed enrichment in immune reactivity, cytokine-chemokine activity, and extracellular matrix. Inhibition of DDR1 enhanced intratumoral infiltration of CD45+, CD8+, and CD3+ immune cells, accompanied by a decrease in the abundance of immune-suppressive cells, suggesting that gliomas exploit DDR1-mediated mechanisms to promote immunosuppression and facilitate tumor growth. We hypothesize that DDR1 in glioma impedes immune surveillance by promoting collagen fiber alignment, as evaluated using collagen-specific second-harmonic generation microscopy. Furthermore, analysis of human datasets demonstrated a negative correlation between DDR1 expression and CD8, suggesting DDR1’s role as a key regulator of immune surveillance in gliomas. Interestingly, when DDR1 knockdown gliomas were implanted in immune deficient mouse models, NSG (NOD-scid-IL2Rγnull) or CD8−/− immune-deficient mice, no significant differences in survival were observed, indicating DDR1 expression in glioma cells dampens anti-glioma immunity. In conclusion, we propose that DDR1 within glioma cells induces an immune-inhibitory TME, reprograms cytokine and chemokine profiles, and promotes collagen fiber alignment, thus inhibiting anti-glioma immunity. Our data highlight DDR1 as a promising therapeutic target, providing a novel avenue for GBM treatment with significant potential.

Abstract 18522: Pyridoxamine (vitamin B6) Mitigation Of Structural Valve Degeneration Of Bioprosthetic Heart Valves

Introduction: Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde treated bovine pericardium (BP) have limited durability due to structural valve degeneration (SVD) resulting from both calcification and advanced glycation end product (AGE) deposition. Hypothesis: We investigated the hypothesis that PYR modification of BHV leaflets mitigates AGE-serum protein uptake, oxidation, and calcification mechanisms in model studies, both in vitro and in vivo. Methods: To model AGE accumulation in BHV, we used well-established in vitro and in vivo models. AGE and serum albumin (SA) uptake in BP were quantitated per immunohistochemistry (IHC) staining intensity. PYR uptake was quantified using a novel fluorescent assay. BP collagen was examined by second-harmonic generation (SHG) microscopy. Calcification studies investigated the activity of BP alkaline phosphatase (ALP), and BP calcium levels were quantitated using a colorimetric assay. In vivo studies used a juvenile rat subdermal BP implantation model with PYR oral administration or BP-PYR modification. Endpoints were calcification, SA, and AGE accumulation per IHC and ALP activity per enzyme histochemistry. Results: PYR modification of BP resulted in high-affinity binding of PYR with 2.0±0.2 μg/mg of PYR binding to BP that was stable for 28 days in physiological conditions. Modification had an inhibitory effect on SA uptake in vitro with 60±5% (p<0.001) reduction in IHC staining intensity; PYR modification of BP resulted in increased protection from oxidative damage in vitro with collagen structure preservation (by SHG) after ten days, while control tissues were degraded. After 28-day of in vivo subdermal BP implantation in rats, a significant 70±5% (p<0.001) decrease and 80±5% (p<0.001) decrease in SA IHC staining and AGE staining, respectively, as well as ALP activity was documented in BP-PYR. Quantitative analysis of calcium content demonstrated a reduction from 108±5 μg/mg in control to 77±5 μg/mg in BP-PYR explants (p<0.001). Conclusions: Our study demonstrated that PYR mitigates SVD mechanisms in vitro and in vivo, limiting both AGE-serum protein accumulation and calcification with associated inhibition of ALP.

Skeletal muscle extracellular matrix structure under applied deformation observed using second harmonic generation microscopy

The mechanical and structural properties of passive skeletal muscle are important for musculoskeletal models in impact biomechanics, rehabilitation engineering and surgical simulation. Passive properties of skeletal muscle depend strongly on the architecture of the extracellular matrix (ECM), but the structure of ECM and its realignment under applied deformation remain poorly understood. We apply second harmonic generation (SHG) microscopy to study muscle ECM in intact muscle samples both under deformation and in the undeformed state. A method for regional relocation was developed, so that the same ECM segment could be viewed before and after applying deformations. Skeletal muscle ECM was viewed at multiple scales and in three states: undeformed, under compression and under tension. Results show that second harmonic generation microscopy provides substantial detail of skeletal muscle ECM over a wide range of length scales, especially the perimysium structure. We present images of individual portions of skeletal muscle ECM both undeformed and subjected to tensile/compressive deformation. We also present data showing the response of the perimysium to a partial thickness cut applied to a section under tensile deformation. Statement of significanceSecond Harmonic Generation (SHG) microscopy is an imaging technique which takes advantage of a non-linear and coherent frequency doubling optical effect that is present in a small number of biological molecules, primarily collagen Type I, II and myosin. Collagen I is the most abundant collagen type in skeletal muscle, making SHG a promising option for visualisation of the skeletal muscle extracellular matrix (ECM). SHG microscopy does not require fixing or staining. This short communication presents the application of SHG microscopy to skeletal muscle ECM to improve our understanding of how collagen fibres reorganise under applied tensile and compression, including microscopic observations of collagen fibre reorganisation for intact samples by using a method to re-identify specific regions in repeated deformation tests.

Quantifying the microstructural and biomechanical changes in the porcine ventricles during growth and remodelling.

Cardiac tissue growth and remodelling (G & R) occur in response to the changing physiological demands of the heart after birth. The early shift to pulmonary circulation produces an immediate increase in ventricular workload, causing microstructural and biomechanical changes that serve to maintain overall physiological homoeostasis. Such cardiac G & R continues throughout life. Quantifying the tissue's mechanical and microstructural changes because of G & R is of increasing interest, dovetailing with the emerging fields of personalised and precision solutions. This study aimed to determine equibiaxial, and non-equibiaxial extension, stress-relaxation, and the underlying microstructure of the passive porcine ventricles tissue at four time points spanning from neonatal to adulthood. The three-dimensional microstructure was investigated via two-photon excited fluorescence and second-harmonic generation microscopy on optically cleared tissues, describing the 3D orientation, rotation and dispersion of the cardiomyocytes and collagen fibrils. The results revealed that during biomechanical testing, myocardial ventricular tissue possessed non-linear, anisotropic, and viscoelastic behaviour. An increase in stiffness and viscoelasticity was noted for the left and right ventricular free walls from neonatal to adulthood. Microstructural analyses revealed concomitant increases in cardiomyocyte rotation and dispersion. This study provides baseline data, describing the biomechanical and microstructural changes in the left and right ventricular myocardial tissue during G & R, which should prove valuable to researchers in developing age-specific, constitutive models for more accurate computational simulations. STATEMENT OF SIGNIFICANCE: There is a dearth of experimental data describing the growth and remodelling of left and right ventricular tissue. The published literature is fragmented, with data reported via different experimental techniques using tissues harvested from a variety of animals, with different gender and ages. This prevents developing a continuum of data spanning birth to death, so limiting the potential that can be leveraged to aid computational modelling and simulations. In this study, equibiaxial, non-equibiaxial, and stress-relaxation data are presented, describing directional-dependent material responses. The biomechanical data is consolidated with equivalent microstructural data, an important element for the development of future material models. Combined, these data describe microstructural and biomechanical changes in the ventricles, spanning G &R from neonatal to adulthood.

Aberration free synthetic aperture second harmonic generation holography.

Second harmonic generation (SHG) microscopy is a valuable tool for optical microscopy. SHG microscopy is normally performed as a point scanning imaging method, which lacks phase information and is limited in spatial resolution by the spatial frequency support of the illumination optics. In addition, aberrations in the illumination are difficult to remove. We propose and demonstrate SHG holographic synthetic aperture holographic imaging in both the forward (transmission) and backward (epi) imaging geometries. By taking a set of holograms with varying incident angle plane wave illumination, the spatial frequency support is increased and the input and output pupil phase aberrations are estimated and corrected - producing diffraction limited SHG imaging that combines the spatial frequency support of the input and output optics. The phase correction algorithm is computationally efficient and robust and can be applied to any set of measured field imaging data.

Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS4) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C6 symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS4 aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.