Second Harmonic Imaging Microscopy Research Articles

Impact of ultrasonic inertial cavitation on the pancreatic tumor stiffness in mice

Pancreatic ductal adenocarcinoma (PDAC) is among the fastest-growing cancers and is predicted to become the second leading cause of cancer-related deaths worldwide by 2030. Ultrasound-induced inertial cavitation (IC) is an emerging therapeutic strategy with promise for treating patients with unresectable tumors. However, the physical impact of IC on tumors remains unclear. In this study, we used orthotopic murine models of pancreatic cancer and employed passive elastography to measure elasticity before and after applying IC to the whole tumor volume. The IC was generated using two focused confocal transducers (center frequency, 1.1 MHz). The output level was adjusted with a real-time feedback loop based on broadband signal acquired with a passive cavitation detector. Cavitation clouds were visualized during treatment using an imaging probe. The results from eight treated mice showed a 24% decrease (p < 0.05) in tumor stiffness after the IC treatment, indicating that the chosen IC sequence can soften the tumor environment. After sacrifice, we utilized second-harmonic imaging microscopy to visualize the collagen network within the tumor, a known contributor to tissue stiffness, and correlated various features of this network (length, width, curvature) with the measured elasticity.

Nonlinear polarization effect of functionalized graphene quantum dots

Graphene quantum dots (GQDs) are nanoscale structures of graphene with quantum properties and edge effects that give photoluminescence properties. The effect of quantum confinement and differences in the nature of GQD structure makes its optical characteristics highly dependent on the size of the structure. This study explains a few exploratory semi-empirical calculations of nonlinear polarization properties of functionalized GQD (fGQD) three-dimensionally. Based on this, the calculation of the linear polarization and first hyperpolarization was performed by the finite field method, which is based on the expansion of the energy and dipole moment. As a result, the fGQD molecule dominantly has high optical nonlinear properties as indicated by the high β values (71 to 4488 a.u.). In general, the first hyperpolarizabilities have a linear relationship with the dipole moments. It was potentially used for the second harmonic imaging microscopy (SHIM) application.

Ultrasound shear wave elastography in pediatric stricturing small bowel Crohn disease: correlation with histology and second harmonic imaging microscopy.

Preclinical animal as well as small exploratory ex vivo and in vivo human studies have suggested that bowel wall shear wave speed (SWS) measurements may be a noninvasive biomarker of intestinal damage. To evaluate the relationships between bowel wall stiffness, measured using ultrasound shear wave elastography (SWE), and intestinal fibrosis and smooth muscle hypertrophy as determined by (1) histology and (2) second harmonic imaging microscopy (SHIM) in surgically resected ileal strictures from pediatric Crohn disease patients. Nineteen pediatric Crohn disease patients with symptomatic ileal strictures underwent research ultrasound examinations before surgical resection between December 2017 and September 2020. Two-dimensional SWE was performed through the area of the most severe stenosis, with measurements obtained from the bowel wall at the 9:00, 12:00 and 3:00 o'clock locations with 0%, 10% and 20% abdominal strain. Overall right lower quadrant stiffness also was documented. Median bowel wall and overall right lower quadrant SWS measurements were correlated with bowel wall histological scores of inflammation, fibrosis and smooth muscle proliferation as well as SHIM collagen signal. Diagnostic ultrasound SWE imaging was obtained from 18 participants. The median age was 16.8 years. There were negative correlations between histological mucosal active inflammation and both bowel wall SWS with 10% abdominal strain (r=-0.50, P = 0.04) and overall right lower quadrant SWS with 20% abdominal strain (r=-0.69, P = 0.002). There were positive correlations between histological muscularis propria inner layer smooth muscle hypertrophy and both median bowel wall SWS with 10% abdominal strain (r = 0.72, P = 0.002) and overall right lower quadrant SWS with 20% abdominal strain (r = 0.71, P = 0.002). There were no associations between ultrasound stiffness metrics and bowel wall SHIM collagen measurements. Bowel wall and overall right lower quadrant ultrasound stiffness measurements correlate with mucosal active inflammation and muscularis propria smooth muscle hypertrophy in pediatric stricturing ileal Crohn disease, but not with intestinal fibrosis.

All-Atom Quantum Mechanical Calculation of the Second-Harmonic Generation of Fluorescent Proteins.

Fluorescent proteins (FPs) are biotags of choice for second-harmonic imaging microscopy (SHIM). Because of their large size, computing their second-harmonic generation (SHG) response represents a great challenge for quantum chemistry. In this contribution, we propose a new all-atom quantum mechanics methodology to compute SHG of large systems. This is now possible because of two recent implementations: the tight-binding GFN2-xTB method to optimize geometries and a related version of the simplified time-dependent density functional theory (sTD-DFT-xTB) to evaluate quadratic response functions. In addition, a new dual-threshold configuration selection scheme is introduced to reduce the computational costs while retaining overall similar accuracy. This methodology was tested to evaluate the SHG of the proteins iLOV and bacteriorhodopsin (bR). In the case of bR, quantitative agreement with respect to experiment was reached for the out-of-resonance low-energy part of the βHRS frequency dispersion. This work paves the way toward an accurate prediction of the SHG of large structures-a requirement for the design of new and improved SHIM biotags.

Ultrafast Charge-Transfer Dynamics in Twisted MoS2/WSe2 Heterostructures.

Two-dimensional transition metal dichalcogenides offer a fascinating platform for creating van der Waals heterojunctions with exciting physical properties. Because of their typical type-II band alignment, photoexcited electrons and holes can separate via interfacial charge transfer. Furthermore, the relative crystallographic alignment of the individual layers in these heterostructures represents an important degree of freedom. Based on both effects, various fascinating ideas for applications in optoelectronics and valleytronics have been suggested. Despite its utmost importance for the design and efficiency of potential devices, the nature and the dynamics of ultrafast charge transfer are not yet well understood. This is mainly because the charge transfer can be surprisingly fast, usually faster than the temporal resolution of previous experimental approaches. Here, we apply time- and polarization-resolved second-harmonic imaging microscopy to investigate the charge-transfer dynamics for three MoS2/WSe2 heterostructures with different stacking angles at a previously unattainable time resolution of ≈10 fs. For 1.70 eV excitation energy, electron transfer from WSe2 to MoS2 is found to depend considerably on the stacking angle with the fastest transfer time observed to be as short as 12 fs. At 1.85 eV excitation energy, ultrafast hole transfer from MoS2 to hybridized states at the Γ-point and to the K-points of WSe2 has to be considered. Surprisingly, the corresponding decay dynamics show only a minor stacking-angle dependence indicating that radiative recombination of momentum-space indirect Γ-K excitons becomes the dominant decay route for all samples.

Tacrolimus action pathways in an ointment base for hypertrophic scar prevention in a rabbit ear model

BackgroundTacrolimus is used to prevent unaesthetic scars due to its action on fibroblast activity and collagen production modulation. ObjectivesTo evaluate the action pathways, from the histopathological point of view and in cytokine control, of tacrolimus ointment in the prevention of hypertrophic scars. MethodsTwenty-two rabbits were submitted to the excision of two 1-cm fragments in each ear, including the perichondrium. The right ear received 0.1% and 0.03% tacrolimus in ointment base twice a day in the upper wound and in the lower wound respectively. The left ear, used as the control, was treated with petrolatum. After 30 days, collagen fibers were evaluated using special staining, and immunohistochemistry analyses for smooth muscle actin, TGF-β and VEGF were performed. ResultsThe wounds treated with 0.1% tacrolimus showed weak labeling and a lower percentage of labeling for smooth muscle actin, a higher proportion of mucin absence, weak staining, fine and organized fibers for Gomori's Trichrome, strong staining and organized fibers for Verhoeff when compared to controls. The wounds treated with 0.03% tacrolimus showed weak labeling for smooth muscle actin, a higher proportion of mucin absence, strong staining for Verhoeff when compared to the controls. There was absence of TGF-β and low VEGF expression. Study limitationsThe analysis was performed by a single pathologist. Second-harmonic imaging microscopy was performed in 2 sample areas of the scar. ConclusionsBoth drug concentrations were effective in suppressing TGF-β and smooth muscle actin, reducing mucin, improving the quality of collagen fibers, and the density of elastic fibers, but only the higher concentration influenced elastic fiber organization.

Tailoring the assembly of collagen fibers in alginate microspheres

The application of microspheres instead of bulk hydrogels in cell-laden biomaterials offers multiple advantages such as a high surface-to-volume-ratio and, consequently, a better nutrition and oxygen transfer to and from cells. The preparation of inert alginate microspheres is facile, quick, and well-established and the fabrication of alginate-collagen microspheres has been previously reported. However, no detailed characterization of the collagen fibrillogenesis in the alginate matrix is available. We use second-harmonic imaging microscopy reflection confocal microscopy and turbidity assay to study the assembly of collagen in alginate microspheres. We show that the assembly of collagen fibers in a gelled alginate matrix is a complex process that can be aided by addition of small polar molecules, such as glycine and by a careful selection of the gelling buffer used to prepare alginate hydrogels.

Second-harmonic imaging microscopy for time-resolved investigations of transition metal dichalcogenides

Two-dimensional transition metal dichalcogenides (TMD) have shown promise for various applications in optoelectronics and so-called valleytronics. Their operation and performance strongly depend on the stacking of individual layers. Here, optical second-harmonic generation in imaging mode is shown to be a versatile tool for systematic time-resolved investigations of TMD monolayers and heterostructures in consideration of the material’s structure. Large sample areas can be probed without the need of any mapping or scanning. By means of polarization dependent measurements, the crystalline orientation of monolayers or the stacking angles of heterostructures can be evaluated for the whole field of view. Pump-probe experiments then allow to correlate observed transient changes of the second-harmonic response with the underlying structure. The corresponding time-resolution is virtually limited by the pulse duration of the used laser. As an example, polarization dependent and time-resolved measurements on mono- and multilayer MoS2 flakes grown on a SiO2/ Si(001) substrate are presented.

Structure Tensors for Dispersed Fibers in Soft Materials

Soft tissues, such as skin, myocardium, and chordae tendineae, typically display anisotropic mechanical behavior due to their fibrous nature. In constitutive modeling, fiber families frequently are assumed to be unidirectional. Recent numerical results, however, display the need to incorporate dispersion of fiber orientation. This evidence gets supplemented by new experimental results based on high-resolution second-harmonic imaging microscopy. Generalized structure-tensor (GST) models are frequently utilized to model fiber dispersion, as they are mathematically easy to treat and demand only a little effort to implement. They can be regarded as Taylor-series expansions of the numerically more challenging angular-integration (AI) method, which encompasses a distribution of fiber orientations together with the associated fiber stress. In this work, we show how low-order GST models give rise to numerical instabilities as they show strong sensitivity with regards to the mean fiber orientation. To overcome these instabilities, we propose a different class of GST models, termed squared GST (SGST), which computes faster, is easier to implement, and converges to the AI faster than previous GST models of similar order. The SGST models promise to be adaptable to generalized problems, such as functional decomposition of fiber density as well as coupling between different fiber families. Advanced simulations with the proposed models will shed light on the complex behavior of fiber reinforced soft materials.

Dynamic Structural Effects on the Second-Harmonic Generation of Tryptophane-Rich Peptides and Gramicidin A.

Peptide chains can model endogenous biotags for applications in second-harmonic imaging microscopy. Such structures are flexible which may strongly affect their structure-property relationship. Here, we explore quantum-mechanically the conformational space of a set of tryptophan-rich model peptides. This has become feasible because of the recently proposed meta-dynamics method based on efficient tight-binding (TB) calculations. The TB version of the simplified time-dependent density functional theory (sTD-DFT-xTB) method is used to evaluate the first hyperpolarizability (β). These new tools enable us to calculate nonlinear optical properties for systems with several thousand atoms and/or to screen large structure ensembles. First, we show that the indole chromophore in tryptophan residues dominates the β response of these systems. Their relative orientation mostly determines the global β tensor and affects the static β response. The results underline the importance of finding low-energy conformers for modeling β of flexible molecules. Additionally, we compare calculated and extrapolated experimental static β. The sTD-DFT-xTB method is capable of providing reliable second-harmonic generation values for tryptophan-rich systems at a fraction of the computational cost of the commonly used TD-DFT/TD-HF levels of theory.

Detection of structural changes in the cell membrane induced by electroporation with second-harmonic imaging microscopy

Detection of structural changes in the cell membrane induced by electroporation with second-harmonic imaging microscopy

Directional ultrafast charge transfer in a WSe2/MoSe2 heterostructure selectively probed by time-resolved SHG imaging microscopy.

Heterostructures of two-dimensional transition metal dichalcogenides (TMD) have shown promise for various optoelectronic and novel valleytronic applications. Due to their type-II band alignment, photoexcited electrons and holes can separate into different layers through ultrafast charge transfer. While this charge-transfer process is critical for potential applications, the underlying mechanisms still remain elusive. Here, we demonstrate for a rotationally mismatched WSe2/MoSe2 heterostructure that directional ultrafast charge transfer between the layers becomes accessible by time-resolved optical second-harmonic generation. By tuning the photon energy of the pump pulse, one of the two materials is resonantly excited, whereas the polarization of the probe pulse can be optimized to selectively detect the charge transfer into the other material. This allows us to explore the interlayer hole transfer from the WSe2 into the MoSe2 layer and vice versa, which appears within a few hundred femtoseconds via hybridized intermediate states at the Γ-point. Our approach enables systematic investigations of the charge transfer in dependence of the rotational layer mismatch in TMD heterostructures.

Nonlinear-response properties in a simplified time-dependent density functional theory (sTD-DFT) framework: Evaluation of the first hyperpolarizability.

Recent developments in nonlinear imaging microscopy show the need to implement new theoretical tools, which are able to characterize nonlinear optical properties in an efficient way. For second-harmonic imaging microscopy (SHIM), quantum chemistry could play an important role to design new exogenous dyes with enhanced first hyperpolarizabilities or to characterize the response origin in large endogenous biological systems. Such methods should be able to screen a large number of compounds while reproducing their trends and to treat large systems in reasonable computation times. To fulfill these requirements, we present a new simplified time-dependent density functional theory (sTD-DFT) implementation to evaluate the first hyperpolarizability where the Coulomb and exchange integrals are approximated by short-range damped Coulomb interactions of transition density monopoles. For an ultra-fast computation of the first hyperpolarizability, a tight-binding version (sTD-DFT-xTB) is also proposed. In our implementation, a sTD-DFT calculation is more than 600 time faster with respect to a regular TD-DFT treatment, while the xTB version speeds up the entire calculation further by at least two orders of magnitude. We challenge our implementation on three test cases: typical push-pull π-conjugated compounds, fluorescent proteins, and a collagen model, which were selected to model requirements for SHIM applications.

Observation of Quasi-Two-Dimensional Polar Domains and Ferroelastic Switching in a Metal, Ca3Ru2O7

Polar domains arise in insulating ferroelectrics when free carriers are unable to fully screen surface-bound charges. Recently discovered binary and ternary polar metals exhibit broken inversion symmetry coexisting with free electrons that might be expected to suppress the electrostatic driving force for domain formation. Contrary to this expectation, we report the first direct observation of polar domains in single crystals of the polar metal Ca3Ru2O7. By a combination of mesoscale optical second-harmonic imaging and atomic-resolution scanning transmission electron microscopy, the polar domains are found to possess a quasi-two-dimensional slab geometry with a lateral size of ∼100 μm and thickness of ∼10 nm. Electronic structure calculations show that the coexistence of electronic and parity-lifting orders arise from anharmonic lattice interactions, which support 90° and 180° polar domains in a metal. Using in situ transmission electron microscopy, we also demonstrate a strain-tuning route to achieve ferroelastic switching of polar metal domains.

Second-harmonic imaging microscopy for identifying colorectal intraepithelial neoplasia.

In this study, second-harmonic imaging microscopy was used to monitor precancerous colorectal lesions at different stages. It was found that the morphology of glands and lamina propria in mucosa changes with the progression of colorectal diseases from normal to low-grade intraepithelial neoplasia to high-grade intraepithelial neoplasia and this microscopy has the ability of direct visualization of these warning symptoms. Furthermore, two morphologic variables were quantified to determine the changes of glands and collagen in lamina propria during the development of colorectal intraepithelial neoplasia. These results suggest that second-harmonic imaging microscopy has the potential in label-freely and effectively distinguishing between normal and precancerous colorectal tissues, and will be helpful for early diagnosis and treatment of colorectal diseases.

A new mode of contrast in biological second harmonic generation microscopy

Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature.

Viewing Cancer in a New Light: Multimodal Label-Free Specific Chemical Imaging to Distinguish Healthy Cell Tissue from Invasive Carcinoma

Microscopy has always been an important tool when determining whether a biopsy contains invasive carcinomas. For this purpose, light microscopy is often used due to its simplicity in construction and operation. The downside to this is the time lost in preparing biopsies before imaging. Typically, the process of obtaining a biopsy, molding, sectioning, mounting and staining of the biopsy on microscopy glass slides and finally imaging the biopsy takes between 6-10 days. If the preparatory steps could be eliminated, valuable time would be freed. Coherent anti-Stokes Raman Scattering (CARS) microscopy offers a possible solution to this obstacle. CARS is a non-linear optical microscopy technique that allows label-free specific imaging of chemical moieties present in a sample. Specifically, lipids (rich in CH2-moieties) and protein (rich in CH-moieties), as well as water, are easily imaged using CARS. A system capable of performing CARS microscopy will generally also be capable of doing Second-Harmonic Imaging Microscopy (SHIM). This technique uses second-harmonic generation (SHG) to achieve frequency doubling of the incident light. SHIM enables imaging of biological structures, which possess the characteristics of being both highly polarizable and ordered on medium to long length scales. Specifically collagen, which is present especially in the basement membrane, is very capable of producing a second-harmonic signal. By combining CARS, SHIM and multi-photon excitation of auto-fluorescent molecules, it is possible to acquire multimodal micrographs of epithelial tissue samples. The CARS measurements gives a unique insight into cell organization in tissue by clearly visualizing the cell membrane bilayer and lipid distribution, while the SHIM reveals the location of the basement membrane and underlying connecting tissue. Our results show that it is possible, in an unlabeled sample, to differentiate between healthy tissue samples and samples that display varying degrees of dysplasia or invasive carcinomas. This opens up for the possibility for future fast bedside cancer diagnostics using CARS microscopy.

Intense Signal Modulation of Nonlinear Optical Scattering and Multiphoton Fluorescence by Ultrasound Irradiation

Tagging linear fluorescence signals by ultrasound has been proposed to achieve improved spatial resolution at known imaging depths. An alternative way to enhance imaging resolution is the use of nonlinear optical methods such as second-harmonic imaging microscopy and two-photon excitation fluorescence. Extending further along these lines, we have studied the fundamental aspects of the effect of ultrasound irradiation on two nonlinear optical effects. In contrast to the focused transducers that are traditionally used in the study of ultrasound -mediated fluorescence that cause a small fluorescence modulation, we have made use of a simple, flat disc-shaped transducer to generate standing waves in the sample cell. Upon ultrasound irradiation, a prompt increase of the hyper-Rayleigh scattering signal with 35% was observed. The signal enhancement shows a clear correlation with the standing wave pattern, as characterized by laser beam deflection scanning measurements. The viability of different possible mechani...

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.

Second Harmonic Imaging of Membrane Potential.

The non-linear optical effect known as second harmonic generation (SHG) has been recognized since the earliest days of the laser. But it has only been in the last 20 years that it has begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. This is because only small modifications are required to equip a standard laser scanning 2-photon microscope for second harmonic imaging microscopy (SHIM). SHG signals from certain membrane-bound dyes are highly sensitive to membrane potential, indicating that SHIM may become a valuable probe of cell physiology. However, for the current generation of dyes and microscopes, the small signal size limits the number of photons that can be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the ability to image neuronal electrical activity with SHIM.