Recovering the susceptibility of antibiotic-resistant bacteria using photooxidative damage.

Multidrug-resistant bacteria are one of the most serious threats to infection control. Few new antibiotics have been developed; however, the lack of an effective new mechanism of their action has worsened the situation. Photodynamic inactivation (PDI) can break antimicrobial resistance, since it potentiates the effect of antibiotics, and induces oxidative stress in microorganisms through the interaction of light with a photosensitizer. This paper addresses the application of PDI for increasing bacterial susceptibility to antibiotics and helping in bacterial persistence and virulence. The effect of photodynamic action on resistant bacteria collected from patients and bacteria cells with induced resistance in the laboratory was investigated. Staphylococcus aureus resistance breakdown levels for each antibiotic (amoxicillin, erythromycin, and gentamicin) from the photodynamic effect (10 µM curcumin, 10 J/cm2) and its maintenance in descendant microorganisms were demonstrated within five cycles after PDI application. PDI showed an innovative feature for modifying the degree of bacterial sensitivity to antibiotics according to dosages, thus reducing resistance and persistence of microorganisms from standard and clinical strains. We hypothesize a reduction in the degree of antimicrobial resistance through photooxidative action combats antibiotic failures.

Label-free drug interaction screening via Raman microscopy.

Development of a simple, label-free screening technique capable of precisely and directly sensing interaction-in-solution over a size range from small molecules to large proteins such as antibodies could offer an important tool for researchers and pharmaceutical companies in the field of drug development. In this work, we present a thermostable Raman interaction profiling (TRIP) technique that facilitates low-concentration and low-dose screening of binding between protein and ligand in physiologically relevant conditions. TRIP was applied to eight protein-ligand systems, and produced reproducible high-resolution Raman measurements, which were analyzed by principal component analysis. TRIP was able to resolve time-depending binding between 2,4-dinitrophenol and transthyretin, and analyze biologically relevant SARS-CoV-2 spike-antibody interactions. Mixtures of the spike receptor-binding domain with neutralizing, nonbinding, or binding but nonneutralizing antibodies revealed distinct and reproducible Raman signals. TRIP holds promise for the future developments of high-throughput drug screening and real-time binding measurements between protein and drug.

Breaking down antibiotic resistance in methicillin-resistant Staphylococcus aureus: Combining antimicrobial photodynamic and antibiotic treatments.

The widespread use of antibiotics drives the evolution of antimicrobial-resistant bacteria (ARB), threatening patients and healthcare professionals. Therefore, the development of novel strategies to combat resistance is recognized as a global healthcare priority. The two methods to combat ARB are development of new antibiotics or reduction in existing resistances. Development of novel antibiotics is a laborious and slow-progressing task that is no longer a safe reserve against looming risks. In this research, we suggest a method for reducing resistance to extend the efficacious lifetime of current antibiotics. Antimicrobial photodynamic therapy (aPDT) is used to generate reactive oxygen species (ROS) via the photoactivation of a photosensitizer. ROS then nonspecifically damage cellular components, leading to general impairment and cell death. Here, we test the hypothesis that concurrent treatment of bacteria with antibiotics and aPDT achieves an additive effect in the elimination of ARB. Performing aPDT with the photosensitizer methylene blue in combination with antibiotics chloramphenicol and tetracycline results in significant reductions in resistance for two methicillin-resistant Staphylococcus aureus (MRSA) strains, USA300 and RN4220. Additional resistant S. aureus strain and antibiotic combinations reveal similar results. Taken together, these results suggest that concurrent aPDT consistently decreases S. aureus resistance by improving susceptibility to antibiotic treatment. In turn, this development exhibits an alternative to overcome some of the growing MRSA challenge.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Hyper-Raman spectroscopy of biomolecules.

The hyper-Raman scattering (HRS) spectra of biologically significant molecules (D-glucose, L-alanine, L-arabinose, L-tartaric acid) in aqueous solutions are reported. The HRS spectra were measured using a picosecond laser at 532 nm operating at a MHz repetition rate. High signal to noise spectra were collected with a commercial spectrometer and CCD without resonant, nanoparticle, or surface enhancement. The HRS peak frequencies, relative intensities, band assignments, and depolarization ratios are examined. By comparing HRS to Raman scattering (RS) and infrared absorption spectra we verify that the IR-active vibrational modes of the target molecules are observed in HRS spectra but come with substantially different peak intensities. The HRS of the biomolecules as well as water, dimethyl sulfoxide, methanol, and ethanol were deposited into a data repository to support the development of theoretical descriptions of HRS for these molecules. Depositing the spectra in a repository also supports future dual detection RS, HRS microscopes which permit simultaneous high-spatial-resolution vibrational spectroscopy of IR-active and Raman-active vibrational modes.

Spectral resolution enhancement for impulsive stimulated Brillouin spectroscopy by expanding pump beam geometry.

Brillouin microscopy has recently emerged as a powerful tool for mechanical property measurements in biomedical sensing and imaging applications. Impulsive stimulated Brillouin scattering (ISBS) microscopy has been proposed for faster and more accurate measurements, which do not rely on stable narrow-band lasers and thermally-drifting etalon-based spectrometers. However, the spectral resolution of ISBS-based signal has not been significantly explored. In this report, the ISBS spectral profile has been investigated as a function of the pump beam's spatial geometry, and novel methodologies have been developed for accurate spectral assessment. The ISBS linewidth was found to consistently decrease with increasing pump-beam diameter. These findings provide the means for improved spectral resolution measurements and pave the way to broader applications of ISBS microscopy.

Neu(t)ralMC: energy-efficient open source Monte Carlo algorithm for assessing photon transport in turbid media.

Light propagation in turbid mediums such as atmosphere, fluids, and biological tissues is a challenging problem which necessitates accurate simulation techniques to account for the effects of multiple scattering. The Monte Carlo method has long established itself as a gold standard and is widely adopted for simulating light transport, however, its computationally intensive nature often requires significant processing power and energy consumption. In this paper a novel, open source Monte Carlo algorithm is introduced which is specifically designed for use with energy-efficient processors, effectively addressing those challenges, while maintaining the accuracy/compatibility and outperforming existing solutions. The proposed implementation optimizes photon transport simulations by exploiting the unique capabilities of Apple's low-power, high-performance M-family of chips. The developed method has been implemented in an open-source software package, enabling seamless adaptation of developed algorithms for specific applications. The accuracy and performance are validated using comprehensive comparison with existing solvers commonly used for biomedical imaging. The results demonstrate that the new algorithm achieves comparable accuracy levels to those of existing techniques while significantly reducing computational time and energy consumption.

Spatial multiplexing for robust optical vortex transmission with optical nonlinearity.

Optical vortex beams, with phase singularity characterized by a topological charge (TC), introduces a new dimension for optical communication, quantum information, and optical light manipulation. However, the evaluation of TCs after beam propagation remains a substantial challenge, impeding practical applications. Here, we introduce vortices in lateral arrays (VOILA), a novel spatial multiplexing approach that enables simultaneous transmission of a lateral array of multiple vortices. Leveraging advanced learning techniques, VOILA effectively decodes TCs, even in the presence of strong optical nonlinearities simulated experimentally. Notably, our approach achieves substantial improvements in single-shot bandwidth, surpassing single-vortex scheme by several orders of magnitude. Furthermore, our system exhibits precise fractional TC recognition in both linear and nonlinear regimes, providing possibilities for high-bandwidth communication. The capabilities of VOILA promise transformative contributions to optical information processing and structured light research, with significant potential for advancements in diverse fields.

High-Dose Photodynamic Therapy Increases Tau Protein Signals in Drosophila.

Amyloid-Detection and imaging of amyloid-ß plaques (Aß) has been a focus in the field of neurodegeneration (ND) due to the high correlation with Parkinson's and Alzheimer's diseases. Here, a novel approach is being proposed and developed to induce and assess those diseases. Photodynamic therapy (PDT) is applied to the fruit fly Drosophila melanogaster as a model of systemic oxidative stress to induce rapid Aß accumulation. Excised brains are evaluated by Brillouin-Raman spectroscopy and microscopy with UV surface emissions (MUSE) to interrogate physical property changes due to fixation and high-dose PDT. MUSE reveals reasonable autofluorescence in the spectral range of Aß, particularly for females, with increased signal once stained. A presence of significant mechanical changes in fresh brains treated with PDT compared to healthy controls is revealed using Brillouin spectroscopy. Aß plaque presence was confirmed with confocal analysis, with female PDT flies yielding nearly four-fold the mean intensity of controls, thus marking PDT as a potential neurodegenerative disease model. MUSE may serve as a viable early screening method for Aß presence and quantification in a research setting. This reduces the time for sample preparation and drastically decreases the cost of Aß quantification.

What is cooking in your kitchen: seeing "invisible" with time-resolved coherent anti-Stokes Raman spectroscopy.

Cooking oil is a critical component of human food and its main component, lipid, is influential to health, but assessing its authenticity and quality can be challenging due to its complex chemical composition. In this study, we introduce a novel application of time-resolved coherent anti-Stokes Raman scattering (T-CARS) spectroscopy for detecting adulteration and understanding the mechanisms of lipid oxidation in various cooking oils. Our research surpasses the limitations of conventional spontaneous Raman spectroscopy, demonstrating that intra-molecular interactions from unsaturated bonds in triglycerides significantly influence vibrational dephasing time. We observed that these dephasing times, although diverse initially, converge to a similar value after heating cycles. Notably, a longer vibrational dephasing of the CH2 symmetric stretching mode was found to correlate with a higher lipid oxidation rate. These findings underscore the potential of T-CARS in identifying and characterizing subtle molecular interactions, offering a transformative approach to understanding molecular dynamics. This research paves the way for broader applications of T-CARS across fields such as chemistry, biomedicine, and material science, marking a significant advancement in the development of innovative analytical techniques.

Enhanced Chemical Sensing with Multiorder Coherent Raman Scattering Spectroscopic Dephasing.

Molecular vibrational spectroscopy is widely used in various sensing and imaging applications, providing intrinsic information at the molecular level. Nonlinear optical interactions using ultrashort laser pulses facilitate the selective coherent excitation of molecular vibrational modes by focusing energy into specific molecular bonds, boosting the signal level for multiple orders of magnitude. The dephasing of such coherence, which is susceptible to the local molecular environment, however, is often neglected. The unique capability of vibrational dephasing dynamics to serve as a unique probe for complex molecular interactions and the effect of local nano- and microenvironments are beyond the reach of conventional, intensity-based spectroscopy. Here, we developed a novel multiorder coherent Raman spectroscopy platform with a special focus on the temporal evolution of molecular vibrational dephasing, termed as time-resolved coherent Raman scattering (T-CRS) spectroscopy. By utilizing a high dynamic range detection, molecular vibrational dynamics and the environmental effects are demonstrated with multidimensional spectroscopic sensing, which promises a new range of applications in biology, materials, and chemical sciences.

En route to nanoscopic quantum optical imaging: counting emitters with photon-number-resolving detectors.

The fundamental understanding of biological pathways requires minimally invasive nanoscopic optical resolution imaging. Many approaches to high-resolution imaging rely on localization of single emitters, such as fluorescent molecules or quantum dots. Additionally, the exact determination of the number of such emitters in an imaging volume is essential for a number of applications; however, in standard intensity-based microscopy it is not possible to determine the number of individual emitters within a diffraction limited spot without initial knowledge of system parameters. Here we explore how quantum measurements of the emitted photons using photon number resolving detectors can be used to address this challenging task. In the proposed new approach, the problem of counting emitters reduces to the task of determining differences between the emitted photon distribution and the Poisson limit. We show that quantum measurements of the number of photons emitted from an ensemble of emitters enable the determination of both the number of emitters and the probability of emission. This method can be applied for any type of single-photon emitters. The scaling laws of this new approach are presented by the Cramer-Rao Lower Bounds, and this technique has great potential in quantum optical imaging with nanoscopic resolution.

Technologically feasible quasi-edge states and topological Bloch oscillation in the synthetic space.

The dimensionality of a physical system is one of the major parameters defining its physical properties. The recently introduced concept of synthetic dimension has made it possible to arbitrarily manipulate the system of interest and harness light propagation in different ways. It also facilitates the transformative architecture of system-on-a-chip devices enabling far reaching applications such as optical isolation. In this report, a novel architecture based on dynamically-modulated waveguide arrays with the Su-Schrieffer-Heeger configuration in the spatial dimension is proposed and investigated with an eye on a practical implementation. The propagation of light through the one-dimensional waveguide arrays mimics time evolution of the field in a synthetic two-dimensional lattice. The addition of the effective gauge potential leads to an exotic topologically protected one-way transmission along adjacent boundary. A cosine-shape isolated band, which supports the topological Bloch oscillation in the frequency dimension under the effective constant force, appears and is localized at the spatial boundary being robust against small perturbations. This work paves the way to improved light transmission capabilities under topological protections in both spatial and spectral regimes and provides a novel platform based on a technologically feasible lithium niobate platform for optical computing and communication.

Segmentation of laser induced retinal lesions using deep learning (December 2021).

OBJECTIVE: Detection of retinal laser lesions is necessary in both the evaluation of the extent of damage from high power laser sources, and in validating treatments involving the placement of laser lesions. However, such lesions are difficult to detect using Color Fundus cameras alone. Deep learning-based segmentation can remedy this, by highlighting potential lesions in the image. METHODS: A unique database of images collected at the Air Force Research Laboratory over the past 30 years was used to train deep learning models for classifying images with lesions and for subsequent segmentation. We investigate whether transferring weights from models that learned classification would improve performance of the segmentation models. We use Pearson's correlation coefficient between the initial and final training phases to reveal how the networks are transferring features. RESULTS: The segmentation models are able to effectively segment a broad range of lesions and imaging conditions. CONCLUSION: Deep learning-based segmentation of lesions can effectively highlight laser lesions, making this a useful tool for aiding clinicians.

Multi-wavelength excitation Brillouin spectroscopy.

We propose and demonstrate, first on simulated spectra and then experimentally, a novel approach to correct the undesired background distortions in the Brillouin spectra caused by molecular filter's absorption, fluorescent emission, ambient room light or any other constant contaminant. The developed multi-wavelength excitation Brillouin spectroscopy method computationally reconstructs the pure Brillouin component of the signal from multiple Brillouin spectra acquired using different excitation wavelengths. By removing the baseline distortions, the approach improves the goodness of fit of the Brillouin peaks, enabling accurate Brillouin shift and linewidth measurements from a wide range of challenging samples. In the present report, we explain the principle behind the method on a set of simulated spectra and present experimental application on an intentionally strongly-distorted spectrum. Utilizing the multi-excitation Brillouin spectroscopy approach, we successfully reconstruct Brillouin spectra of a highly-scattering sample, initially rendered not analyzable by excessive iodine absorption and contamination by out-of-focus light.

Coherent anti-Stokes Raman scattering microspectroscopy: an emerging technique for non-invasive optical assessment of a local bio-nano-environment.

Raman spectroscopy provides a non-invasive, chemically-specific optical imaging of biological objects without relying on endogenous labels. Nonlinear Raman spectroscopy allows non-invasive imaging at much faster speed with an improved spatial resolution and axial sectioning capability. In this report we propose a novel use of nonlinear Raman spectroscopy as a sensor of local nano-environment. Time-resolved coherent anti-Stokes Raman spectrograms are found to be sensitive to small variations of local structural changes, which are not normally observed using conventional Raman spectroscopy.

New insights into a hydrogen bond: hyper-Raman spectroscopy of DMSO-water solution.

Hydrogen bonding plays an essential role in biological processes by stabilizing proteins and lipid structures as well as controlling the speed of enzyme catalyzed reactions. Dimethyl sulfoxide-water (DMSO-H2O) solution serves as a classical model system by which the direct and indirect effects of hydrogen bonding between water hydrogens and the sulfoxide functional group can be explored. The complex transition from self-bonding to heterogeneous bonding is important, and multiple spectroscopic approaches are needed to provide a detailed assessment of those interactions. In this report, for the first time, hyper-Raman scattering was successfully employed to investigate molecular interactions in DMSO-H2O system. We measured the improper blueshift of the C-S and C-H stretching modes of DMSO caused by partial charge transfer and enhanced bond polarization. By detecting differences in the frequency shifts of C-S and C-H modes for low DMSO concentrations (<33 mol%) we find evidence of the intermolecular bonds between water and the DMSO methyl groups. We exploit the high sensitivity of hyper-Raman scattering to the low frequency librations of H2O to observe a change in librational mode population providing insight into existing questions about the coordination of H2O around DMSO molecules and the formation of the H2O shell around DMSO molecules proposed in prior simulation studies. These results demonstrate that hyper-Raman spectroscopy can be a practical spectroscopic technique to study the intermolecular bonding of model systems and test claims about model system bonding generated by theoretical calculations.

Enhanced coupling of light into a turbid medium through microscopic interface engineering.

There are many optical detection and sensing methods used today that provide powerful ways to diagnose, characterize, and study materials. For example, the measurement of spontaneous Raman scattering allows for remote detection and identification of chemicals. Many other optical techniques provide unique solutions to learn about biological, chemical, and even structural systems. However, when these systems exist in a highly scattering or turbid medium, the optical scattering effects reduce the effectiveness of these methods. In this article, we demonstrate a method to engineer the geometry of the optical interface of a turbid medium, thereby drastically enhancing the coupling efficiency of light into the material. This enhanced optical coupling means that light incident on the material will penetrate deeper into (and through) the medium. It also means that light thus injected into the material will have an enhanced interaction time with particles contained within the material. These results show that, by using the multiple scattering of light in a turbid medium, enhanced light-matter interaction can be achieved; this has a direct impact on spectroscopic methods such as Raman scattering and fluorescence detection in highly scattering regimes. Furthermore, the enhanced penetration depth achieved by this method will directly impact optical techniques that have previously been limited by the inability to deposit sufficient amounts of optical energy below or through highly scattering layers.

Eye safety implications of high harmonic generation in zinc selenide.

Polycrystalline zinc selenide (ZnSe) has been the subject of many nonlinear optics studies for wavelengths under 4.0 µm including sum/difference frequency generation, harmonic generation, and filamentation. In this report, the conversion efficiency of high harmonic generation (HHG) in ZnSe is quantified for mid-infrared wavelengths ranging from 2.7 µm to 8.0 µm. By increasing the fundamental wavelength, we demonstrate that HHG in thick ZnSe targets is limited by the band gap. The high conversion efficiency of mid-infrared to near-infrared light in ZnSe raises concerns of a nonlinear retinal hazard. We contrast the HHG behavior of ZnSe against the observed harmonic generation of calcium fluoride, BK7, and fused silica over the same wavelengths.

Highly efficient tunable picosecond deep ultraviolet laser system for Raman spectroscopy.

We present a narrowband laser system tunable from 219 to 236 nm for deep ultraviolet (DUV) Raman spectroscopy. The demonstrated laser system produces 6.7 ps nearly transform-limited pulses with energy up to 0.36 µJ at 100 kHz repetition rate. The system consists of a two-stage optical parametric amplifier (OPA) of a narrowband continuous wave diode laser and subsequent frequency conversion to the DUV radiation. We achieve more than 300 mW in the signal wave using ${{\rm LiB}_3}{{\rm O}_5}$LiB3O5 (LBO) and ${{\rm BaB}_2}{{\rm O}_4}$BaB2O4 (BBO) crystals, with the total 2.7 W pump after the two-stage OPA. We reach 12% conversion efficiency of the OPA signal wave into the DUV radiation using type-I phase matching in the BBO crystal. Finally, we demonstrate the applicability of the system for DUV Raman spectroscopy by collecting a high dynamic range, high spectral resolution spontaneous Raman spectrum of air.