3D Hydrogel Culture System Recapitulates Key Tuberculosis Phenotypes and Demonstrates Pyrazinamide Efficacy.

The mortality caused by tuberculosis (TB) infections is a global concern, and there is a need to improve understanding of the disease. Current in vitro infection models to study the disease have limitations such as short investigation durations and divergent transcriptional signatures. This study aims to overcome these limitations by developing a 3D collagen culture system that mimics the biomechanical and extracellular matrix (ECM) of lung microenvironment (collagen fibers, stiffness comparable to in vivo conditions) as the infection primarily manifests in the lungs. The system incorporates Mycobacterium tuberculosis (Mtb) infected human THP-1 or primary monocytes/macrophages. Dual RNA sequencing reveals higher mammalian gene expression similarity with patient samples than 2D macrophage infections. Similarly, bacterial gene expression more accurately recapitulates in vivo gene expression patterns compared to bacteria in 2D infection models. Key phenotypes observed in humans, such as foamy macrophages and mycobacterial cords, are reproduced in the model. This biomaterial system overcomes challenges associated with traditional platforms by modulating immune cells and closely mimicking in vivo infection conditions, including showing efficacy with clinically relevant concentrations of anti-TB drug pyrazinamide, not seen in any other in vitro infection model, making it reliable and readily adoptable for tuberculosis studies and drug screening.

Harmonic to anharmonic tuning of moiré potential leading to unconventional Stark effect and giant dipolar repulsion in WS2/WSe2 heterobilayer.

Excitonic states trapped in harmonic moiré wells of twisted heterobilayers is an intriguing testbed for exploring many-body physics. However, the moiré potential is primarily governed by the twist angle, and its dynamic tuning remains a challenge. Here we demonstrate anharmonic tuning of moiré potential in a WS2/WSe2 heterobilayer through gate voltage and optical power. A gate voltage can result in a local in-plane perturbing field with odd parity around the high-symmetry points. This allows us to simultaneously observe the first (linear) and second (parabolic) order Stark shift for the ground state and first excited state, respectively, of the moiré trapped exciton - an effect opposite to conventional quantum-confined Stark shift. Depending on the degree of confinement, these excitons exhibit up to twenty-fold gate-tunability in the lifetime (100 to 5 ns). Also, exciton localization dependent dipolar repulsion leads to an optical power-induced blueshift of ~ 1 meV/µW - a five-fold enhancement over previous reports.

Pulsed Carrier Gas Assisted High-Quality Synthetic 3R-Phase Sword-like MoS2: A Versatile Optoelectronic Material.

Synthesizing a material with the desired polymorphic phase in a chemical vapor deposition (CVD) process requires a delicate balance among various thermodynamic variables. Here, we present a methodology to synthesize rhombohedral (3R)-phase MoS2 in a well-defined sword-like geometry having lengths up to 120 µm, uniform width of 2-3 µm and thickness of 3-7 nm by controlling the carrier gas flow dynamics from continuous mode to pulsed mode during the CVD growth process. Characteristic signatures such as high degree of circular dichroism (∼58% at 100 K), distinct evolution of low-frequency Raman peaks and increasing intensity of second harmonic signals with increasing number of layers conclusively establish the 3R-phase of the material. A high value (∼844 pm/V) of second-order susceptibility for few-layer-thick MoS2 swords signifies the potential of MoS2 to serve as an atomically thin nonlinear medium. A field effect mobility of 40 cm2/V-s and Ion/Ioff ratio of ∼106 further confirm the electronic-grade standard of this 3R-phase MoS2. These findings are significant for the development of emerging quantum electronic devices utilizing valley-based physics and nonlinear optical phenomena in layered materials.

Focus-engineered sub-diffraction imaging in infrared-sensitive third-order sum frequency generation microscope.

We experimentally demonstrate sub-diffraction imaging in infrared-sensitive third-order sum frequency generation (TSFG) microscope using focal-field engineering technique. The TSFG interaction studied here makes use of two mid infrared photons and a single 1040 nm pump photon to generate up-converted visible photons. Focal field engineering scheme is implemented using a Toraldo-style single annular phase mask imprinted on the 1040 nm beam using a spatial light modulator. The effect of focal field engineered excitation beam on the non-resonant-TSFG process is studied by imaging isolated silicon sub-micron disks and periodic grating structures. Maximum reduction in the measured TSFG central-lobe size by ∼43% with energy in the central lobe of 35% is observed in the presence of phase mask. Maximum contrast improvement of 30% is observed for periodic grating structures. Furthermore, to validate the infrared sensitivity of the focus engineered TSFG microscope, we demonstrate imaging of amorphous Germanium-based guided-mode resonance structures, and polystyrene latex beads probed near the O-H vibrational region. We also demonstrate the utility of the focus engineered TSFG microscope for high resolution imaging of two-dimensional layered material. Focus-engineered TSFG process is a promising imaging modality that combines infrared selectivity with improved resolution and contrast, making it suitable for nanostructure and surface layer imaging.

Strong near band-edge excited second-harmonic generation from multilayer 2H Tin diselenide.

We report strong second-harmonic generation (SHG) from 2H polytype of multilayer Tin diselenide (SnSe2) for fundamental excitation close to the indirect band-edge in the absence of excitonic resonances. Comparison of SHG and Raman spectra from exfoliated SnSe2 flakes of different polytypes shows strong (negligible) SHG and Raman Eg mode at 109 cm-1 (119 cm-1), consistent with 2H (1T) polytypes. The difference between the A1g-Eg Raman peak positions is found to exhibit significant thickness dependent for the 1T form, which is found to be absent for the 2H form. The observed thickness dependence of SHG with rapid oscillations in signal strength for small changes in flake thickness are in good agreement with a nonlinear wave propagation model considering nonlinear polarization with alternating sign from each monolayer. The nonlinear optical susceptibility extracted from SHG signal comparison with standard quartz samples for 1040 nm excitation is found to be more than 4-times higher than that at 1550 nm. This enhanced nonlinear response at 1040 nm is attributed to the enhanced nonlinear optical response for fundamental excitation close to the indirect band-edge. We also study SHG from heterostructures of monolayer MoS2/multilayer SnSe2 which allows us to unambiguously compare the nonlinear optical response of SnSe2 with MoS2. We find the SHG signal and any interference effect in the overlap region to be dominated by the SnSe2 layer for the excitation wavelengths considered. The comparison of SHG from SnSe2 and MoS2 underscores that the choice of the 2D material for a particular nonlinear optical application is contextual on the wavelength range of interest and its optical properties at those wavelengths. The present works further highlights the usefulness of near band-edge enhancement of nonlinear processes in emerging 2D materials towards realizing useful nanophotonic devices.

A simple fabrication strategy for orientationally accurate twisted heterostructures.

Van der Waals (vdW) heterostructure is a type of metamaterial where multiple layers of 2D materials are vertically aligned at controlled misorientation. The relative rotation in between the adjacent layers, or the twist angle between them plays a crucial role in changing the electronic band structure of the superlattice. The assembly of multi-layers of precisely twisted two dimensional layered materials requires knowledge of the atomic structure at the edge of the flake. It may be artificially created by the 'tear and stack' process. Otherwise, the crystallographic orientation needs to be determined through invasive processes such as transmission electron microscopy or scanning tunneling microscopy, and via second-harmonic generation (SHG). Here, we demonstrate a simple and elegant transfer protocol using only an optical microscope as a edge identifier tool through which, controlled transfer of twisted homobilayer and heterobilayer transition metal dichalcogenides is performed with close to 100% yield. The fabricated twisted vdW heterostructures have been characterized by SHG, Raman spectroscopy and photoluminiscence spectroscopy, confirming the desired twist angle within ∼0.5° accuracy. The presented method is reliable, quick and prevents the use of invasive tools which is desirable for reproducible device functionalities.

Beam steering in a narrow-beam phosphor down-converted white light visible light communication link using transmitter lens decentering.

Indoor visible light communication (VLC) systems with narrow beams can achieve practical few-meters-long wireless optical links. Such links can operate at low power levels and high data rate for supporting point-to-point or multipoint communication. The narrow-beam VLC links can, however, benefit from beam steering to support mobility of user equipment and cater to multiple users. Simple beam-steering techniques with minimal changes to the existing optical hardware are required to enable widespread adoption of beam steering in VLC links. We study the performance of a simple transmitter-lens-decenter-based beam-steering scheme in a VLC link, utilizing a phosphor down-converted blue laser transmitter. The beam-steering angle and hence the receiver coverage depend on the transmitter lens decenter and the choice of the transmitter and receiver lens's focal lengths. Optical ray tracing is used to quantify the collection efficiency achievable with beam steering, choose a suitable receiver lens, and understand the role of off-axis aberration in the system performance. In our experimental implementation, the transmitter lens decentering technique results in a maximum steering angle of 7.1°. This corresponds to a receiver coverage of 30 cm per cm of transmitter lens decenter for a fixed link length of 300 cm. The measured on-axis white light color coordinates of (0.286, 0.253) is found to shift toward warmer white colors with beam steering. The on-axis illuminance level of ∼19lux decreases slightly with beam steering and is found to be below the maximum permissible exposure limit for indoor illumination. We also quantify the data communication performance as a function of beam steering using on-off modulated data. Bit-error rates below the forward error correction limit are obtained for receiver coverage diameter of 75 cm and 60 cm for 1.25 Gbps and 1.5 Gbps data rates, respectively.

Quantitative probe for in-plane piezoelectric coupling in 2D materials.

Piezoelectric response in two-dimensional (2D) materials has evoked immense interest in using them for various applications involving electromechanical coupling. In most of the 2D materials, piezoelectricity is coupled along the in-plane direction. Here, we propose a technique to probe the in-plane piezoelectric coupling strength in layered nanomaterials quantitively. The method involves a novel approach for in-plane field excitation in lateral Piezoresponse force microscopy (PFM) for 2D materials. Operating near contact resonance has enabled the measurement of the piezoelectric coupling coefficients in the sub pm/V range. Detailed methodology for the signal calibration and the background subtraction when PFM is operated near the contact resonance of the cantilever is also provided. The technique is verified by estimating the in-plane piezoelectric coupling coefficients (d11) for freely suspended MoS2 of one to five atomic layers. For 2D-MoS2 with the odd number of atomic layers, which are non-centrosymmetric, finite d11 is measured. The measurements also indicate that the coupling strength decreases with an increase in the number of layers. The techniques presented would be an effective tool to study the in-plane piezoelectricity quantitatively in various materials along with emerging 2D-materials.

A biphasic response of polymerized Type 1 collagen architectures to dermatan sulfate.

Collagen I, the most abundant extracellular matrix (ECM) protein in vertebrate tissues provides mechanical durability to tissue microenvironments and regulates cell function. Its fibrillogenesis in biological milieu is predominantly regulated by dermatan sulfate proteoglycans, proteins conjugated with iduronic acid-containing dermatan sulfate (DS) glycosaminoglycans (GAG). Although DS is known to regulate tissue function through its modulation of Coll I architecture, a precise understanding of the latter remains elusive. We investigated this problem by visualizing the fibrillar pattern of fixed Coll I gels polymerized in the presence of varying concentrations of DS using second harmonic generation microscopy. Measuring mean second harmonic generation signal (which estimates the ordering of the fibrils), and surface occupancy (which estimates the space occupied by fibrils) supported by confocal reflectance microscopy, our observations indicated that the effect on fibril pattern of DS is contextual upon the latter's concentrations: Lower levels of DS resulted in sparse disorganized fibrils; higher levels restore organization, with fibrils occupying greater space. An appropriate change in elasticity as a result of DS levels was also observed through atomic force microscopy. Examination of dye-based GAG staining and scanning electron microscopy suggested distinct constitutions of Coll I gels when polymerized with higher and lower levels of DS. We observed that adhesion of the invasive ovarian cancer cells SKOV3 decreased for lower DS levels but was partially restored at higher DS levels. Our study shows how the Coll I gel pattern-tuning of DS is of relevance for understanding its biomaterial applications and possibly, pathophysiological functions.

Polarization independent enhancement of zeroth order diffracted second harmonic from multilayer gallium selenide on a silicon resonant metasurface.

We demonstrate polarization-independent resonant-enhancement of second harmonic generation (SHG) from multilayer Gallium Selenide (GaSe) on a silicon-based resonant metasurface. Two-dimensional hexagonal photonic lattice with circularly symmetric silicon meta-atoms are designed to achieve resonant field enhancement at the fundamental wavelength independent of the incident polarization direction. Such structures are however found to exhibit strong resonant field depolarization effects at the fundamental excitation fields resulting in modified nonlinear polarization components when compared to the native GaSe layer. Furthermore, the sub-wavelength metasurface designed to have resonances at the fundamental wavelengths act as a higher order diffraction grating at the second harmonic wavelength. Nonlinear wave propagation simulations show that the higher order diffracted SHG exhibit strong polarization dependent enhancement with characteristics very different from the native GaSe layer. In this context, polarization independent enhancement of the second harmonic signal is achieved only for the zeroth order diffracted component. Experimental study of second harmonic generation from the GaSe layer integrated with the silicon metasurface shows maximum nonlinear signal enhancement on-resonance with polarization dependence identical to the native GaSe layer by selectively detecting the zeroth-order diffracted component. This work shows that it is not sufficient to use symmetric meta-atoms in such 2D material integrated resonant metasurfaces for achieving polarization independent nonlinear optical enhancement. Depolarization of the resonant fields and higher-order diffraction at the nonlinear signal wavelength need to be considered as well.

Evolution of high-frequency Raman modes and their doping dependence in twisted bilayer MoS2.

Twisted van der Waals heterostructures provide a new platform for studying strongly correlated quantum phases. The interlayer coupling in these heterostructures is sensitive to the twist angle (θ) and key to controllably tuning several interesting properties. Here, we demonstrate the systematic evolution of the interlayer coupling strength with twist angle in bilayer MoS2 using a combination of Raman spectroscopy and classical simulations. At zero doping, we observe a monotonic increase in the separation between the A1g and E2g1 mode frequencies as θ decreases from 10°â†’ 1°, and the separation approaches that of a bilayer at small twist angles. Furthermore, using doping dependent Raman spectroscopy, we reveal the θ dependent softening and broadening of the A1g mode, whereas the E2g1 mode remains unaffected. Using first principles based simulations, we demonstrate large (weak) electron-phonon coupling for the A1g (E2g1) mode, which explains the experimentally observed trends. Our study provides a non-destructive way to characterize the twist angle and the interlayer coupling and establishes the manipulation of phonons in twisted bilayer MoS2 (twistnonics).

Nonlinear Optics in Dielectric Guided-Mode Resonant Structures and Resonant Metasurfaces.

Nonlinear optics is an important area of photonics research for realizing active optical functionalities such as light emission, frequency conversion, and ultrafast optical switching for applications in optical communication, material processing, precision measurements, spectroscopic sensing and label-free biological imaging. An emerging topic in nonlinear optics research is to realize high efficiency optical functionalities in ultra-small, sub-wavelength length scale structures by leveraging interesting optical resonances in surface relief metasurfaces. Such artificial surfaces can be engineered to support high quality factor resonances for enhanced nonlinear optical interaction by leveraging interesting physical mechanisms. The aim of this review article is to give an overview of the emerging field of nonlinear optics in dielectric based sub-wavelength periodic structures to realize efficient harmonic generators, wavelength mixers, optical switches etc. Dielectric metasurfaces support the realization of high quality-factor resonances with electric field concentrated either inside or in the vicinity of the dielectric media, while at the same time operate at high optical intensities without damage. The periodic dielectric structures considered here are broadly classified into guided-mode resonant structures and resonant metasurfaces. The basic physical mechanisms behind guided-mode resonances, electromagnetically-induced transparency like resonances and bound-states in continuum resonances in periodic photonic structures are discussed. Various nonlinear optical processes studied in such structures with example implementations are also reviewed. Finally, some future directions of interest in terms of realizing large-area metasurfaces, techniques for enhancing the efficiency of the nonlinear processes, heterogenous integration, and extension to non-conventional wavelength ranges in the ultra-violet and infrared region are discussed.

Third-harmonic generation in multilayer Tin Diselenide under the influence of Fabry-Perot interference effects.

Two-dimensional layered materials are in general known to exhibit strong layer dependent nonlinear optical response owing to the crystal symmetry and associated phase matching considerations. Here we report up-conversion of 1550 nm incident light using third-harmonic generation (THG) in multilayered tin di-selenide (SnSe2) and study its thickness dependence by simultaneously acquiring spatially-resolved images in the forward and backward propagation direction. We find good agreement between the experimental measurements and a coupled-wave equation model we have developed when including the effect of Fabry-Perot interference between the SnSe2 layer and the surrounding medium. We extract the magnitude of the third order electronic nonlinear optical susceptibility of SnSe2, for the first time to our knowledge, by comparing its nonlinear response with a glass substrate and find this to be ∼1500 times higher than that of glass. We also study the polarization dependence and find good agreement with the expected angular dependence of nonlinear polarization considering the crystal symmetry of SnSe2. The large nonlinear optical susceptibility of multi-layer SnSe2 makes it a promising material for studying nonlinear optical effects. This work demonstrates that in addition to the large inherent nonlinear optical susceptibility, the high refractive index of these materials and optical absorption above the bandgap strongly influence the overall nonlinear optical response and its thickness dependence characteristics.

Iduronate-2-Sulfatase-Regulated Dermatan Sulfate Levels Potentiate the Invasion of Breast Cancer Epithelia through Collagen Matrix.

Cancer epithelia show elevation in levels of sulfated proteoglycans including dermatan sulfates (DS). The effect of increased DS on cancer cell behavior is still unclear. We hypothesized that decreased expression of the enzyme Iduronate-2-sulfatase (IDS) can lead to increased DS levels, which would enhance the invasion of cancer cells. Breast cancer sections shows depleted IDS levels in tumor epithelia, when compared with adjacent untransformed breast tissues. IDS signals showed a progressive decrease in the non-transformed HMLE, transformed but non-invasive MCF-7 and transformed and invasive MDA-MB-231 cells, respectively, when cultured on Type 1 collagen scaffolds. DS levels measured by ELISA increased in an inverse-association with IDS levels. Knockdown of IDS in MCF-7 epithelia also increased the levels of DS. MCF-7 cells with depleted IDS expression, when imaged using two photon-excited fluorescence and second harmonic generation microscopy, exhibited a mesenchymal morphology with multiple cytoplasmic projections compared with epithelioid control cells, interacted with their surrounding matrix, and showed increased invasion through Type 1 collagen matrices. Both these traits were phenocopied when control MCF-7 cells were cultivated on Type 1 collagen gels polymerized in the presence of DS. In monolayer cultures, DS had no effect on MCF-7 migration. In the context of our demonstration that DS enhances the elastic modulus of Type 1 collagen gels, we propose that a decrease of IDS expression leads to accumulation within cancer epithelia of DS: the latter remodels the collagen around cancer cells leading to changes in cell shape and invasiveness through fibrillar matrix milieu.

Strong Single- and Two-Photon Luminescence Enhancement by Nonradiative Energy Transfer across Layered Heterostructure.

The strong light-matter interaction in monolayer transition metal dichalcogenides (TMDs) is promising for nanoscale optoelectronics with their direct band gap nature and the ultrafast radiative decay of the strongly bound excitons these materials host. However, the impeded amount of light absorption imposed by the ultrathin nature of the monolayers impairs their viability in photonic applications. Using a layered heterostructure of a monolayer TMD stacked on top of strongly absorbing, nonluminescent, multilayer SnSe2, we show that both single-photon and two-photon luminescence from the TMD monolayer can be enhanced by a factor of 14 and 7.5, respectively. This is enabled through interlayer dipole-dipole coupling induced nonradiative Förster resonance energy transfer (FRET) from SnSe2 underneath, which acts as a scavenger of the light unabsorbed by the monolayer TMD. The design strategy exploits the near-resonance between the direct energy gap of SnSe2 and the excitonic gap of monolayer TMD, the smallest possible separation between donor and acceptor facilitated by van der Waals heterojunction, and the in-plane orientation of dipoles in these layered materials. The FRET-driven uniform single- and two-photon luminescence enhancement over the entire junction area is advantageous over the local enhancement in quantum dot or plasmonic structure integrated 2D layers and is promising for improving quantum efficiency in imaging, optoelectronic, and photonic applications.

Tissue Thickness Effects on Immunohistochemical Staining Intensity of Markers of Cancer.

High-quality patient samples are required for reliable immunohistochemistry test outcomes that provide a significant benefit for patient care. Among the preanalytic variables in tissue handling, tissue thickness is thought to be easily controlled; however, whether the thickness of the tissue effects the staining intensity for antibody immunohistochemistry has not been quantitatively demonstrated. To investigate, we cut multiblock tissue sections of tonsil, liver, and kidney at 2, 4, 6, and 8 µm thicknesses. Interferometry measurements of the sectioned paraffin showed a <1 µm variation within a preset microtome thickness. Sections were then immunostained with antibodies targeting different cellular localizations; Ki-67 and BCL6 (nuclear), CD7 (membranous), and cytokeratin (cytoplasmic). A pathologist annotated regions of interest for each marker and performed brightfield and whole-slide visual scoring. Then a pixel-wise processing algorithm determined intensity of each pixel in these regions of interest and binned them into predetermined 0, 1+, 2+, or 3+ intensities. Visual scores from brightfield and whole-slide images were highly correlated to the percentage of pixels in each intensity bin. A stepwise increase was observed in pathologist scores and algorithmically defined percentage of pixels in each bin with increasing thickness demonstrating that changes in preset section thickness impacts staining intensity. The use of tissue thickness outside vendors' recommendations might change the intensity including the proportion of positive and negative cells and eventually the overall diagnosis outcome. Therefore, we recommend that tissue be consistently cut within the middle of thickness range specified by the assay manufacturer.

Microscopic study of resonant third-harmonic generation from amorphous silicon nanodisk arrays.

A detailed microscopic study of third-harmonic generation (THG) from two-dimensional arrays of sub-wavelength spaced amorphous silicon nanodisks is reported. The arrays are designed to support broadband, minimally angle-sensitive resonances for the fundamental excitation wavelength in the 1500 nm region. This results in resonantly enhanced visible THG in the green spectral range with ∼500-fold enhancement on-resonance, compared to the unpatterned a-Si thin-film. THG multispectral microscopic imaging reveals individual nanodisks with enhanced nonlinear signal on-resonance. For increasing pump intensities, spatially dependent saturation effects are observed for the first time, to the best of our knowledge, in such dielectric nanostructure arrays with THG images showing a reversal of contrast.

Mapping molecular orientation with phase sensitive vibrationally resonant sum-frequency generation microscopy.

We demonstrate a phase sensitive, vibrationally resonant sum-frequency generation (PSVR-SFG) microscope that combines high resolution, fast image acquisition speed, chemical selectivity, and phase sensitivity. Using the PSVR-SFG microscope, we generate amplitude and phase images of the second-order susceptibility of collagen I fibers in rat tail tendon tissue on resonance with the methylene vibrations of the protein. We find that the phase of the second-order susceptibility shows dependence on the effective polarity of the fibril bundles, revealing fibrous collagen domains of opposite orientations within the tissue. The presence of collagen microdomains in tendon tissue may have implications for the interpretation of the mechanical properties of the tissue.

Rapid vibrational imaging with sum frequency generation microscopy.

We demonstrate rapid vibrational imaging based on sum frequency generation (SFG) microscopy with a collinear excitation geometry. Using the tunable picosecond pulses from a high-repetition-rate optical parametric oscillator, vibrationally selective imaging of collagen fibers is achieved with submicrometer lateral resolution. We furthermore show simultaneous SFG and second harmonic generation imaging to emphasize the compatibility of the microscope with other nonlinear optical modalities.

Multiplicative and subtractive focal volume engineering in coherent Raman microscopy.

Rigorous calculations are performed to study the effective reduction of the nonlinear excitation volumes when using phase-only masks to condition the pump and Stokes driving fields. Focal volume reduction was achieved using both a multiplicative operation of the excitation fields as well as a subtractive operation. Using a tunable optical bottle beam for the Stokes field, an effective reduction of the width of the excitation volume by a factor of 1.5 can be achieved in the focal plane. Further reduction of the focal volume introduces a rapid growth of sidelobes, which renders such volumes unsuitable for imaging applications. In addition, phase sensitive detection was found to provide information from selective sub-divisions of the engineered coherent anti-Stokes Raman scattering excitation volume. In the case of isolated nanoparticles, an apparent resolution improvement by a factor of 3 is demonstrated, and it is shown that the size of sub-diffraction-limited particles can be accurately determined using phase sensitive detection.