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The emerging technique of mid-infrared optical coherence tomography (MIR-OCT) takes advantage of the reduced scattering of MIR light in various materials and devices, enabling tomographic imaging at deeper penetration depths. Because of challenges in MIR detection technology, the image acquisition time is, however, significantly longer than for tomographic imaging methods in the visible/near-infrared. Here we demonstrate an alternative approach to MIR tomography with high-speed imaging capabilities. Through femtosecond nondegenerate two-photon absorption of MIR light in a conventional Si-based CCD camera, we achieve wide-field, high-definition tomographic imaging with chemical selectivity of structured materials and biological samples in mere seconds.

A comprehensive theoretical analysis of photoinduced forces in an illuminated nanojunction, formed between an atomic force microscopy tip and a sample, is presented. The formalism is valid within the dipolar approximation and includes multiple scattering effects between the tip, sample, and a planar substrate through a dyadic Green's function approach. This physically intuitive description allows a detailed look at the quantitative contribution of multiple scattering effects to the measured photoinduced force, effects that are typically unaccounted for in simpler analytical models. Our findings show that the presence of the planar substrate and anisotropy of the tip have a substantial effect on the magnitude and the spectral response of the photoinduced force exerted on the tip. Unlike previous models, our calculations predict photoinduced forces that are within range of experimentally measured values in photoinduced force microscopy (PiFM) experiments.

We examine the mechanical eigenmodes of a quartz tuning fork (QTF) for the purpose of facilitating its use as a probe for multifrequency atomic force microscopy (AFM). We perform simulations based on the three-dimensional finite element method and compare the observed motions of the beams with experimentally measured resonance frequencies of two QTF systems. The comparison enabled us to assign the first seven asymmetric eigenmodes of the QTF. We also find that a modified version of single beam theory can be used to guide the assignment of mechanical eigenmodes of QTFs. The usefulness of the QTF for multifrequency AFM measurements is demonstrated through photoinduced force microscopy measurements. By using the QTF in different configurations, we show that the vectorial components of the photoinduced force can be independently assessed and that lateral forces can be probed in true noncontact mode.

We perform time-resolved pump-probe microscopy measurements by recording the local force between a sharp tip and the photo-excited sample as a readout mechanism for the material's nonlinear polarization. We show that the photo-induced force is sensitive to the same excited state dynamics as measured in an optical pump-probe experiment. Ultrafast pump-probe force microscopy constitutes a non-optical detection technique with nanoscale resolution that pushes pump-probe sensitivities close to the realm of single molecule studies.

We demonstrate the utility of picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis to rapidly map lipophilic components in cardiovascular tissues, facilitating the interrogation of atherosclerosis. © 2011 OSA: BODA/NTM/OMP/OTA.

An overview is presented of recent trends in coherent anti-Stokes Raman scattering (CARS) microscopy. We briefly discuss the influence of tissue scattering on the CARS signal, methods for controlling the CARS emission and prospects for surface-enhancement of the CARS radiation.

CARS microscopy is used to quantitatively investigate the process of skin optical clearing. Using glycerol and dimethyl-sulfoxide as the clearing agents, we find that tissue scattering is a highly nonlinear function of agent concentration. © 2007 Optical Society of America.

Two-photon absorption in indirect gap semiconductors is a frequently encountered, but not well-understood phenomenon. To address this, the real-density matrix approach is applied to describe two-photon absorption in silicon through the excitonic response to the interacting fields. This approach produces an analytical expression for the dispersion of the two-photon absorption coefficient for indirect-gap materials and can be used to explain trends in reported experimental data for bulk silicon both old and new with minimal fitting., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

Photons do not carry sufficient momentum to induce indirect optical transitions in semiconducting materials, such as silicon, necessitating the assistance of lattice phonons to conserve momentum. Compared to direct bandgap semiconductors, this renders silicon a less attractive material for a wide variety of optoelectronic applications. In this work, we introduce an alternative strategy to fulfill the momentum-matching requirement in indirect optical transitions. We demonstrate that when confined to scales below ∼3 nm, photons acquire sufficient momentum to allow electronic transitions at the band edge of Si without the assistance of a phonon. Confined photons allow simultaneous energy and momentum conservation in two-body photon-electron scattering; in effect, converting silicon into a direct bandgap semiconductor. We show that this less-explored concept of light-matter interaction leads to a marked increase in the absorptivity of Si from the UV to the near-IR. The strategy provides opportunities for more efficient use of indirect semiconductors in photovoltaics, energy conversion, light detection, and emission.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942 cm-1 and a shoulder at 2917 cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870 cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030 cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

The nature of enhanced photoemission in disordered and amorphous solids is an intriguing question. A point in case is light emission in porous and nanostructured silicon, a phenomenon that is still not fully understood. In this work, we study structural photoemission in heterogeneous cross-linked silicon glass, a material that represents an intermediate state between the amorphous and crystalline phases, characterized by a narrow distribution of structure sizes. This model system shows a clear dependence of photoemission on size and disorder across a broad range of energies. While phonon-assisted indirect optical transitions are insufficient to describe observable emissions, our experiments suggest these can be understood through electronic Raman scattering instead. This phenomenon, which is not commonly observed in crystalline semiconductors, is driven by structural disorder. We attribute photoemission in this disordered system to the presence of an excess electron density of states within the forbidden gap (Urbach bridge) where electrons occupy trapped states. Transitions from gap states to the conduction band are facilitated through electron-photon momentum matching, which resembles Compton scattering but is observed for visible light and driven by the enhanced momentum of a photon confined within the nanostructured domains. We interpret the light emission in structured silicon glass as resulting from electronic Raman scattering. These findings emphasize the role of photon momentum in the optical response of solids that display disorder on the nanoscale.

Halide perovskites belong to an important family of semiconducting materials with electronic properties that enable a myriad of applications, especially in photovoltaics and optoelectronics. Their optical properties, including photoluminescence quantum yield, are affected and notably enhanced at crystal imperfections where the symmetry is broken and the density of states increases. These lattice distortions can be introduced through structural phase transitions, allowing charge gradients to appear near the interfaces between phase structures. In this work, we demonstrate controlled multiphase structuring in a single perovskite crystal. The concept uses cesium lead bromine (CsPbBr 3 ) placed on a thermoplasmonic TiN/Si metasurface and enables single-, double-, and triple-phase structures to form on demand above room temperature. This approach promises application horizons of dynamically controlled heterostructures with distinctive electronic and enhanced optical properties.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research has experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

The development and application of nonlinear optical (NLO) microscopy methods in biomedical research have experienced rapid growth over the past three decades. Despite the compelling power of these methods, optical scattering limits their practical use in biological tissues. This tutorial offers a model-based approach illustrating how analytical methods from classical electromagnetism can be employed to comprehensively model NLO microscopy in scattering media. In Part I, we quantitatively model focused beam propagation in non-scattering and scattering media from the lens to focal volume. In Part II, we model signal generation, radiation, and far-field detection. Moreover, we detail modeling approaches for major optical microscopy modalities including classical fluorescence, multi-photon fluorescence, second harmonic generation, and coherent anti-Stokes Raman microscopy.

Alkyne-based Raman tags have proven their utility for biological imaging. Although the alkynyl stretching mode is a relatively strong Raman scatterer, the detection sensitivity of alkyne-tagged compounds is ultimately limited by the magnitude of the probe's Raman response. In order to improve the performance of alkyne-based Raman probes, we have designed several tags that benefit from π-π conjugation as well as from additional n-π conjugation with a sulfur linker. We show that the sulfur linker provides additional enhancement and line width narrowing, offering a simple yet effective strategy for improving alkyne-based Raman tags. We validate the utility of various sulfur-linked alkyne tags for cellular imaging through stimulated Raman scattering microscopy.

Spectral imaging in the mid-infrared (MIR) range provides simultaneous morphological and chemical information of a wide variety of samples. However, current MIR technologies struggle to produce high-definition images over a broad spectral range at acquisition rates that are compatible with real-time processes. We present a novel spectral imaging technique based on nondegenerate two-photon absorption of temporally chirped optical MIR pulses. This approach avoids complex image processing or reconstruction and enables high-speed acquisition of spectral data cubes (xyω) at high-pixel density in under a second.

We demonstrate experimentally the detection of magnetic force at optical frequencies, defined as the dipolar Lorentz force exerted on a photoinduced magnetic dipole excited by the magnetic component of light. Historically, this magnetic force has been considered elusive since, at optical frequencies, magnetic effects are usually overshadowed by the interaction of the electric component of light, making it difficult to recognize the direct magnetic force from the dominant electric forces. To overcome this challenge, we develop a photoinduced magnetic force characterization method that exploits a magnetic nanoprobe under structured light illumination. This approach enables the direct detection of the magnetic force, revealing the magnetic nearfield distribution at the nanoscale, while maximally suppressing its electric counterpart. The proposed method opens up new avenues for nanoscopy based on optical magnetic contrast, offering a research tool for all-optical spin control and optomagnetic manipulation of matter at the nanoscale.

Surface-enhanced coherent anti-Stokes Raman scattering (SE-CARS) takes advantage of surface plasmon resonances supported on metallic nanostructures to amplify the coherent Raman response of target molecules. While these metallic antennas have found significant success in SE-CARS studies, photoinduced morphological changes to the nanoantenna under ultrafast excitation introduce significant hurdles in terms of stability and reproducilibty. These hurdles need to be overcome in order to establish SE-CARS as a reliable tool for rapid biomolecular sensing. Here, we address this challenge by performing molecular CARS measurements enhanced by nanoantennas made from high-index dielectric particles with more favorable thermal properties. We present the first experimental demonstration of enhanced molecular CARS signals observed at Si nanoantennas, which offer much improved thermal stability compared to their metallic counterparts.

Photo-induced force microscopy (PiFM) is a scan probe technique that offers images with spectroscopic contrast at a spatial resolution in the nanometer range. PiFM utilizes the non-propagating, enhanced near field at the apex of a sharp tip to locally induce a polarization in the sample, which in turn produces an additional force acting on the cantilevered tip. This photo-induced force, though in the pN range or less, can be extracted from the oscillation properties of the cantilever, thus enabling the generation of photo-induced force maps. Since its inception in 2010, the PiFM technique has grown into a useful nano-spectrocopic tool that has expanded its reach in terms of imaging capabilities and applications. In this review, we present various technical implementations of the PiFM approach. In addition, we discuss the physical origin of the PiFM signal, highlighting the contributions from dipole-dipole forces as well as forces that derive from photo-thermal processes.

We discuss an experimental configuration consisting of {Au film}-molecule-{Au particle} or {Au film}-molecule-{Si particle} nanojunctions for performing wide-field surface-enhanced CARS (SE-CARS) measurements in a reproducible and controllable manner. While the allowable illumination dosage in the {Au film}-molecule-{Au particle} case is limited by the strong two-photon background from the gold, we successfully generate a detectable coherent Raman response from a molecular monolayer using the lowest reported average power densities to-date. With a vision to minimize the two-photon background and the intrinsic losses observed in all-metal plasmonic systems, we examine the possibility of using high-index dielectric particles on top of a thin metal film to generate strong nanoscopic hotspots. We demonstrate repeatable SE-CARS measurements at the {Au film}-molecule-{Si particle} heterojunction, underlining the usability of this experimental geometry. This work paves the way for the development of next-generation of chemical and biomolecular sensing assays that can minimize some of the major drawbacks encountered in fragile and lossy all-metal plasmonic systems.

Recent work on mid-infrared (MIR) detection through the process of non-degenerate two-photon absorption (NTA) in semiconducting materials has shown that wide-field MIR imaging can be achieved with standard Si cameras. While this approach enables MIR imaging at high pixel densities, the low nonlinear absorption coefficient of Si prevents fast NTA-based imaging at lower illumination doses. Here, we overcome this limitation by using InGaAs as the photosensor. Taking advantage of the much higher nonlinear absorption coefficient of this direct bandgap semiconductor, we demonstrate high-speed MIR imaging up to 500 fps with under 1 ms exposure per frame, enabling 2D or 3D mapping without pre- or post-processing of the image., (© 2021 Author(s).)

Fibrin glue (FG) has potential as a delivery vehicle for photosensitizer directly to the resection cavity, so it may bypass the blood-brain barrier (BBB) and increase the concentration of successfully delivered photosensitizer. A specialized form of photodynamic therapy (PDT), photochemical internalization (PCI), which involves both photosensitizer and chemotherapeutic agent internalization, can locally inhibit the growth of cells. This will allow the reduction of recurrence of malignant gliomas around surgical resection. This study will look at the efficacy of FG loaded with drugs in mediating both PDT and PCI in inhibiting 3-dimensional tumor spheroid growth in vitro. Experiments were conducted on spheroids comprised of F98 glioma cells using photosensitizer AlPcS 2a and chemotherapeutic drug bleomycin (BLM). At 2-, 24-, 48-, and 72-h increments, supernatant covering an FG layer within a well was collected and replaced by fresh medium, then added to spheroid-containing wells, which contained the respective chemicals for PDT and PCI. The wells were then exposed to light treatment from a diode laser, and after, spheroid growth was monitored for a period of 14 days. Significant spheroid growth inhibition was observed in both PDT and PCI modalities, but was far greater in PCI. Additionally, complete growth suppression was achieved via PCI at the highest radiant exposure. Achieving a slow photosensitizer release, significant F98 spheroid inhibition was observed in FG-mediated PDT and PCI. The present study showed BLM-PCI was the most efficacious of the two modalities.

As a key industrial nitrogenous product and a critical environmental pollutant, ammonia broadly affects our daily lives. Rapid and sensitive detection of ammonia is essential to both environmental monitoring and process control for industrial manufacturing. Here, we present a protocol for rapid detection of low amounts of ammonia in the aqueous phase, via surface-enhanced Raman spectroscopy. We believe the mechanism and speed of the approach demonstrate its potential toward applications in operando electrochemical catalysis and in situ ammonia detection. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Significance: Coherent Raman scattering (CRS) microscopy is an optical imaging technique with capabilities that could benefit a broad range of biomedical research studies., Aim: We reflect on the birth, rapid rise, and inescapable growing pains of the technique and look back on nearly four decades of developments to examine where the CRS imaging approach might be headed in the next decade to come., Approach: We provide a brief historical account of CRS microscopy, followed by a discussion of the challenges to disseminate the technique to a larger audience. We then highlight recent progress in expanding the capabilities of the CRS microscope and assess its current appeal as a practical imaging tool., Results: New developments in Raman tagging have improved the specificity and sensitivity of the CRS technique. In addition, technical advances have led to CRS microscopes that can capture hyperspectral data cubes at practical acquisition times. These improvements have broadened the application space of the technique., Conclusion: The technical performance of the CRS microscope has improved dramatically since its inception, but these advances have not yet translated into a substantial user base beyond a strong core of enthusiasts. Nonetheless, new developments are poised to move the unique capabilities of the technique into the hands of more users.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation., Competing Interests: The authors have declared that no competing interests exist.

As a key precursor for nitrogenous compounds and fertilizer, ammonia affects our lives in numerous ways. Rapid and sensitive detection of ammonia is essential, both in environmental monitoring and in process control for industrial production. Here we report a novel and nonperturbative method that allows rapid detection of ammonia at low concentrations, based on the all-optical detection of surface-enhanced Raman signals. We show that this simple and affordable approach enables ammonia probing at selected regions of interest with high spatial resolution, making in situ and operando observations possible., Competing Interests: All authors declare no competing interests., (© 2020 The Authors.)

Investigating the behavior of breast cancer cells via reaction kinetics may help unravel the mechanisms that underlie metabolic changes in tumors. However, obtaining human in vivo kinetic data is challenging because of difficulties associated with measuring these parameters. Nondestructive methods of measuring lipid content in live cells provide a novel approach to quantitatively model lipid synthesis and consumption. In this study, coherent Raman scattering microscopy was used to probe de novo intracellular lipid content. Combining nonlinear optical microscopy and Michaelis-Menten kinetics-based simulations, we isolated fatty acid synthesis/consumption rates and elucidated effects of altered lipid metabolism in T47D breast cancer cells. When treated with 17β-estradiol, the lipid utilization in cancer cells jumped by twofold. Meanwhile, the rate of de novo lipid synthesis in cancer cells treated with 17β-estradiol was increased by 42%. To test the model in extreme metabolic conditions, we treated T47D cells with etomoxir. Our kinetic analysis demonstrated that the rate of key enzymatic reactions dropped by 75%. These results underline the capability to probe lipid alterations in live cells with minimum interruption and to characterize lipid metabolism in breast cancer cells via quantitative kinetic models and parameters., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chemical imaging based on mid-infrared (MIR) spectroscopic contrast is an important technique with a myriad of applications, including biomedical imaging and environmental monitoring. Current MIR cameras, however, lack performance and are much less affordable than mature Si-based devices, which operate in the visible and near-infrared regions. Here, we demonstrate fast MIR chemical imaging through non-degenerate two-photon absorption (NTA) in a standard Si-based charge-coupled device (CCD). We show that wide-field MIR images can be obtained at 100 ms exposure times using picosecond pulse energies of only a few femtojoules per pixel through NTA directly on the CCD chip. Because this on-chip approach does not rely on phase matching, it is alignment-free and does not necessitate complex postprocessing of the images. We emphasize the utility of this technique through chemically selective MIR imaging of polymers and biological samples, including MIR videos of moving targets, physical processes and live nematodes., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Purpose: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC)., Methods: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function., Results: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation., Conclusions: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy., (Copyright © 2020. Published by Elsevier Inc.)

When light illuminates the junction formed between a sharp metal tip and a sample, different mechanisms can contribute to the measured photoinduced force simultaneously. Of particular interest are the instantaneous force between the induced dipoles in the tip and in the sample, and the force related to thermal heating of the junction. A key difference between these 2 force mechanisms is their spectral behavior. The magnitude of the thermal response follows a dissipative (absorptive) Lorentzian line shape, which measures the heat exchange between light and matter, while the induced dipole response exhibits a dispersive spectrum and relates to the real part of the material polarizability. Because the 2 interactions are sometimes comparable in magnitude, the origin of the chemical selectivity in nanoscale spectroscopic imaging through force detection is often unclear. Here, we demonstrate theoretically and experimentally how the light illumination gives rise to the 2 kinds of photoinduced forces at the tip-sample junction in the midinfrared. We comprehensively address the origin of the spectroscopic forces by discussing cases where the 2 spectrally dependent forces are entwined. The analysis presented here provides a clear and quantitative interpretation of nanoscale chemical measurements of heterogeneous materials and sheds light on the nature of light-matter coupling in optomechanical force-based spectronanoscopy.

We describe the operating principle and performance of a recently developed surface plasmon-enhanced optical sensor that utilizes two-photon excited luminescence of a planar gold film as the reporter signal. The sensor enables direct visualization of nanoscopic binding events near a sensing surface. Light is coupled to the Au/sample interface in an objective-based Kretschmann configuration to excite surface plasmon polariton (SPP) modes at a metal-dielectric interface. The gold luminescence induced by the confined optical field between the particle and the film is detected in the epi-direction by a far-field camera where individual binding events show up as diffraction limited bright spots against a dark background. We study the sensor's emission spectrum and the distance dependence between the target and substrate, which both suggest that the optical signal of the sensor originates from electron-hole pair excitations in the planar Au film. In addition, we show that the well-behaved pointspread function of the sensor enables a straightforward implementation of super-resolution techniques. Finally, we demonstrate the utility of the sensor for detecting DNA binding events, underlining the sensor's usefulness for label-free imaging of nanoscopic particles and biomolecular interactions.

We present a study of the optical second-order nonlinearity of type I collagen fibers grown in vitro via second harmonic generation (SHG) experiments and analyze the observed polarization-resolved SHG signal using previously reported SHG analytical expressions obtained for anisotropic tissue. Our results indicate that the effective second-order nonlinearity measured in the grown fibers is one order of magnitude lower than that of native collagen fibers. This is attributed to the formation of loose and dispersive fibrillar networks of thinner collagen fibrils that constitute the reassembled collagen fibers. This is confirmed by scanning electronic microscopy (SEM) imaging and the polarization dependence of the SHG signal. The measured values of the anisotropy parameter ρ of the reassembled collagen fibers are found to be similar to that obtained for native fibers on the relevant sub-µm scale., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Purpose: This study examined whether hyperspectral stimulated Raman scattering (hsSRS) microscopy can detect differences in meibum lipid to protein composition of normal and evaporative dry eye subjects with meibomian gland dysfunction., Methods: Subjects were evaluated for tear breakup time (TBUT), staining, meibum expression and gland dropout. Expressed meibum was analyzed using SRS vibrational signatures in the CH stretching region (2800-3050 cm -1 ). Vertex component analysis and K-means clustering were used to group the spectral signatures into four fractions containing high lipid (G1) to high protein (G4)., Results: Thirty-three subjects could be statistically analyzed using pooled meibum (13 with stable tear films (TBUTs > 10 s) and 20 with unstable tear films (TBUTs ≤ 10 s). Significant differences in meibum from subjects with unstable vs. stable TBUTs were found for the G1 fraction (medians 0.164 and 0.020, respectively; p = 0.012) and the G2 fraction (medians 0.244 and 0.272, respectively; p = 0.045). No differences were observed for the G3 and G4 fractions. Single orifice samples were not significantly different vs. pooled samples from the fellow eye, and eyelid sector samples (nasal, central and temporal) G2:G3 fractional components were not significantly different (p = 0.449). Spearman analysis suggested a significant inverse correlation between G1 fraction and TBUT (R = -0.351; p = 0.045)., Conclusions: hsSRS microscopy allows compositional analysis of expressed meibum from humans which correlated to changes in TBUT. These findings support the hypothesis that hsSRS may be useful in classifying meibum quality and evaluating the effects of therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent work has shown that optical magnetism, generally considered a challenging light-matter interaction, can be significant at the nanoscale. In particular, the dielectric nanostructures that support magnetic Mie resonances are low-loss and versatile optical magnetic elements that can effectively manipulate the magnetic field of light. However, the narrow magnetic resonance band of dielectric Mie resonators is often overshadowed by the electric response, which prohibits the use of such nanoresonators as efficient magnetic nanoantennas. Here, we design and fabricate a silicon (Si) truncated cone magnetic Mie resonator at visible frequencies and excite the magnetic mode exclusively by a tightly focused azimuthally polarized beam. We use photoinduced force microscopy to experimentally characterize the local electric near-field distribution in the immediate vicinity of the Si truncated cone at the nanoscale and then create an analytical model of such structure that exhibits a matching electric field distribution. We use this model to interpret the PiFM measurement that visualizes the electric near-field profile of the Si truncated cone with a superior signal-to-noise ratio and infer the magnetic response of the Si truncated cone at the beam singularity. Finally, we perform a multipole analysis to quantitatively present the dominance of the magnetic dipole moment contribution compared to other multipole contributions into the total scattered power of the proposed structure. This work demonstrates the excellent efficiency and simplicity of our method of using Si truncated cone structure under APB illumination compared to other approaches to achieve dominant magnetic excitations.

We investigate the tip-enhanced thermal expansion force for nanoscale chemical imaging and spectroscopy in the tip-sample junction. It is found, both theoretically and experimentally, that the tip-enhanced absorption of the near-field at the tip followed by sample expansion shows characteristic behaviors with respect to the sample thickness and the incident laser pulse width. The van der Waals interaction plays a major role in exerting a force on the tip from the thermally expanded sample. The force behavior of the photoinduced force microscope (PiFM) is compared with that of the existing photothermal-induced resonance technique (PTIR) to unravel the ambiguous thermal expansion force mechanism. The present study opens up new opportunities for enhancing the performance of optical nanoscopy and spectroscopy such as chemical imaging of nanobiomaterials and the local field mapping of photonic devices, including surface polaritons on van der Waals materials with the assistance of the thermal expansion of a functionalized tip.

We studied the use of vibrationally resonant, third-order sum-frequency generation (TSFG) for imaging of biological samples. We found that laser-scanning TSFG provides vibrationally sensitive imaging capabilities of lipid droplets and structures in sectioned tissue samples. Although the contrast is based on the infrared-activity of molecular modes, TSFG images exhibit a high lateral resolution of 0.5 µ m or better. We observed that the imaging properties of TSFG resemble the imaging properties of coherent anti-Stokes Raman scattering (CARS) microscopy, offering a nonlinear infrared alternative to coherent Raman methods. TSFG microscopy holds promise as a high-resolution imaging technique in the fingerprint region where coherent Raman techniques often provide insufficient sensitivity., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

Molecular surface-enhanced Raman spectra recorded at single plasmonic nanojunctions using a 7 ps pulse train exhibit vibrational up-pumping and population inversion. The process is assigned to plasmon-driven, dark, impulsive electron-vibration (e-v) excitation. Both optical (Raman) pumping and hot-electron mediated excitation can be rejected by the characteristic spectra, which allow the simultaneous measurement of vibrational temperature of the molecules and electronic temperature of the metal. Vibrational populations are determined from anti-Stokes to Stokes intensity ratios, while the electron temperature is obtained from the anti-Stokes branch of the electronic Raman scattering continuum. Population inversion survives in high-frequency vibrations that effectively decouple from the metal.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically. Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR. See related commentary by Commisso and Debnath, p. 800 This article is highlighted in the In This Issue feature, p. 781 ., (©2018 American Association for Cancer Research.)

Label-free nonlinear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD + ) is widely used for high-resolution cellular redox imaging. In this work, we combined three label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: (i) TPEF optical redox ratio (ORR = FAD + /NADH + FAD + ), (ii) coherent Raman scattering of cellular lipids, and (iii) second harmonic generation of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial (PME) cells and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17β-estradiol (E2) increased glycolysis in both PME and T47D ER + breast cancer acini. However, PME cells displayed increased lipid content with no effect on ECM, while T47D cells had decreased lipid storage ( P < 0.001) and significant reorganization of collagen. By measuring deuterated lipids synthesized from exogenously administered deuterium-labeled glucose, treatment of T47D cells with E2 increased both lipid synthesis and consumption rates. These results confirm that glucose is a significant source for the cellular synthesis of lipid in glycolytic breast cancer cells, and that the combination of cellular redox and lipid fraction imaging endpoints is a powerful approach with new and complementary information content. Significance: These findings provide unique insight into metabolic processes, revealing correlations between cancer metastasis and cellular redox state, lipid metabolism, and extracellular matrix. Cancer Res; 78(10); 2503-12. ©2018 AACR ., (©2018 American Association for Cancer Research.)

We describe the development and performance of a new type of optical sensor suitable for registering the binding/dissociation of nanoscopic particles near a gold sensing surface. The method shares similarities with surface plasmon resonance microscopy but uses a completely different optical signature for reading out binding events. This new optical read-out mechanism, which we call confined optical field enhanced fluorescence emission (Cofefe), uses pulsed surface plasmon polariton fields at the gold/liquid interface that give rise to confined optical fields upon binding of the target particle to the gold surface. The confined near-fields are sufficient to induce two-photon absorption in the gold sensor surface near the binding site. Subsequent radiative recombination of the electron-hole pairs in the gold produces fluorescence emission, which can be captured by a camera in the far-field. Bound nanoparticles show up as bright confined spots against a dark background on the camera. We show that the Cofefe sensor is capable of detecting gold and silicon nanoparticles, as well as polymer nanospheres and sub-μm lipid droplets in a label-free manner with average illumination powers of less than 10 μW/μm 2 .

Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero , exerting long-lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western-style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8-12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow-derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at 3 weeks of age, WD-fed mice developed a decreased abundance of Parabacteroides and Lactobacillus , together with increased Ruminococcus and decreased tight junction gene expression by 20 weeks, whereas microbiota of mice exposed to PQQ retained compositional stability with age, which was associated with improved liver health. Conclusion : Exposure to a maternal WD induces early gut dysbiosis and disrupts intestinal tight junctions, resulting in BMDM polarization and induction of proinflammatory and profibrotic programs in the offspring that persist into adulthood. Disrupted macrophage and microbiota function can be attenuated by short-term maternal treatment with PQQ prior to weaning, suggesting that reshaping the early gut microbiota in combination with reprogramming macrophages during early weaning may alleviate the sustained proinflammatory environment, preventing the rapid progression of nonalcoholic fatty liver disease to nonalcoholic steatohepatitis in offspring of obese mothers. ( Hepatology Communications 2018;2:313-328).

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub- μ m resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family., Competing Interests: The authors declare that there are no conflicts of interest related to this article.