A Novel Non-Destructive Rapid Tool for Estimating Amino Acid Composition and Secondary Structures of Proteins in Solution.

Amino-acid protein composition plays an important role in biology, medicine, and nutrition. Here, a groundbreaking protein analysis technique that quickly estimates amino acid composition and secondary structure across various protein sizes, while maintaining their natural states is introduced and validated. This method combines multivariate statistics and the thermostable Raman interaction profiling (TRIP) technique, eliminating the need for complex preparations. In order to validate the approach, the Raman spectra are constructed of seven proteins of varying sizes by utilizing their amino acid frequencies and the Raman spectra of individual amino acids. These constructed spectra exhibit a close resemblance to the actual measured Raman spectra. Specific vibrational modes tied to free amino and carboxyl termini of the amino acids disappear as signals linked to secondary structures emerged under TRIP conditions. Furthermore, the technique is used inversely to successfully estimate amino acid compositions and secondary structures of unknown proteins across a range of sizes, achieving impressive accuracy ranging between 1.47% and 5.77% of root mean square errors (RMSE). These results extend the uses for TRIP beyond interaction profiling, to probe amino acid composition and structure.

Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities.

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

Optical multiband polarimetric modulation sensing for gender and species identification of flying native solitary pollinators.

Native pollinators are crucial to local ecosystems but are under threat with the introduction of managed pollinators, e.g., honeybees (Apis mellifera). We explored the feasibility of employing the entomological lidar technique in native pollinator abundance studies. This study included individuals of both genders of three common solitary bee species, Osmia californica, Osmia lignaria, and Osmia ribifloris, native to North America. Properties including optical cross-section, degree of linear polarization, and wingbeat power spectra at all three wavelengths have been extracted from the insect signals collected by a compact stand-off sensing system. These properties are then used in the classification analysis. For species with temporal and spatial overlapping, the highest accuracies of our method exceed 96% (O. ribifloris & O. lignaria) and 93% (O. lignaria & O. californica). The benefit of employing the seasonal activity and foraging preference information in enhancing identification accuracy has been emphasized.

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.

Non-Destructive Direct Pericarp Thickness Measurement of Sorghum Kernels Using Extended-Focus Optical Coherence Microscopy.

Non-destructive measurements of internal morphological structures in plant materials such as seeds are of high interest in agricultural research. The estimation of pericarp thickness is important to understand the grain quality and storage stability of seeds and can play a crucial role in improving crop yield. In this study, we demonstrate the applicability of fiber-based Bessel beam Fourier domain (FD) optical coherence microscopy (OCM) with a nearly constant high lateral resolution maintained at over ~400 µm for direct non-invasive measurement of the pericarp thickness of two different sorghum genotypes. Whereas measurements based on axial profiles need additional knowledge of the pericarp refractive index, en-face views allow for direct distance measurements. We directly determine pericarp thickness from lateral sections with a 3 µm resolution by taking the width of the signal corresponding to the pericarp at the 1/e threshold. These measurements enable differentiation of the two genotypes with 100% accuracy. We find that trading image resolution for acquisition speed and view size reduces the classification accuracy. Average pericarp thicknesses of 74 µm (thick phenotype) and 43 µm (thin phenotype) are obtained from high-resolution lateral sections, and are in good agreement with previously reported measurements of the same genotypes. Extracting the morphological features of plant seeds using Bessel beam FD-OCM is expected to provide valuable information to the food processing industry and plant breeding programs.

Giving entangled photons new colors.

A fiber-based modulator shifts photon frequency while preserving quantum correlations.

Quantum optical immunoassay: upconversion nanoparticle-based neutralizing assay for COVID-19.

In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2 [Formula: see text]g/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.

Resonant Degenerate Four-Wave Mixing at the Defect Energy Levels of 2D Organic-Inorganic Hybrid Perovskite Crystals.

Two-dimensional organic-inorganic lead halide perovskites are generating great interest due to their optoelectronic characteristics such as high solar energy conversion efficiency and a tunable direct band gap in the visible regime. However, the presence of defect states within the two-dimensional crystal structure can affect these properties, resulting in changes to their band gap emission as well as the emergence of nonlinear optical phenomena. Here, we have investigated the effects of the presence of defect states on the nonlinear optical phenomena of the 2D hybrid perovskite (BA)2(MA)2Pb3Br10. When two pulses, one narrowband pump pulse centered at 800 nm and one supercontinuum pulse with bandwidth from 800-1100 nm, are incident on a perovskite flake, degenerate four-wave mixing (FWM) occurs, with peaks corresponding to the energy levels of the defect states present within the crystal. The longer carrier lifetime of the defect state, in comparison to that of virtual transitions that take place in nonresonant FWM processes, allows for a larger population of electrons to be excited by the second pump photon, resulting in increased FWM signal at the defect energy levels. The quenching of the two-photon luminescence as flake thickness increases is also observed and attributed to the increased presence of defects within the flake at larger thicknesses. This technique shows the potential of detecting defect energy levels in crystals using FWM for a variety of optoelectronic applications.

Raman Characterization of Fungal DHN and DOPA Melanin Biosynthesis Pathways.

Fungal melanins represent a resource for important breakthroughs in industry and medicine, but the characterization of their composition, synthesis, and structure is not well understood. Raman spectroscopy is a powerful tool for the elucidation of molecular composition and structure. In this work, we characterize the Raman spectra of wild-type Aspergillus fumigatus and Cryptococcus neoformans and their melanin biosynthetic mutants and provide a rough "map" of the DHN (A. fumigatus) and DOPA (C. neoformans) melanin biosynthetic pathways. We compare this map to the Raman spectral data of Aspergillus nidulans wild-type and melanin biosynthetic mutants obtained from a previous study. We find that the fully polymerized A. nidulans melanin cannot be classified according to the DOPA pathway; nor can it be solely classified according to the DHN pathway, consistent with mutational analysis and chemical inhibition studies. Our approach points the way forward for an increased understanding of, and methodology for, investigating fungal melanins.

Quantum Optical Immunoassay: Upconversion Nanoparticle-based Neutralizing Assay for COVID-19.

In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2 {\mu}g/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.

Simultaneous In Situ Characterizations of Ultrashort Laser Pulses and the Nonlinear Susceptibility of the Irradiated Medium via Time-Resolved Hybrid Coherent Anti-Stokes Raman Scattering Spectroscopy.

We propose and test a method of simultaneously characterizing ultrashort laser pulses and the nonlinear susceptibility of the irradiated medium at the site where nonlinear interactions occur. In this method, a coherent anti-Stoke Raman scattering (CARS) spectrogram is generated with the hybrid CARS technique. We confirm that abundant information is contained in, and can be extracted from, this spectrogram and develop an extraction algorithm. With this method, quantitative and comparable broadband CARS imaging based on phase retrieval is achievable without a second material for nonresonant background generation. Furthermore, this method also paves the way for studying highly localized nonlinear light-matter interactions.

Extended focal depth Fourier domain optical coherence microscopy with a Bessel-beam - LP02 mode - from a higher order mode fiber.

We present a robust fiber-based setup for Bessel-like beam extended depth-of-focus Fourier-domain optical coherence microscopy, where the Bessel-like beam is generated in a higher order mode fiber module. In this module a stable guided LP02 core mode is selectively excited by a long period grating written in the higher order mode fiber. Imaging performance of this system in terms of lateral resolution and depth of focus was analyzed using samples of suspended microbeads and compared to the case where illumination is provided by the fundamental LP01 mode of a single mode fiber. Illumination with the LP02 mode allowed for a lateral resolution down to 2.5 µm as compared to 4.5 µm achieved with the LP01 mode of the single mode fiber. A three-fold enhancement of the depth of focus compared to a Gaussian beam with equally tight focus is achieved with the LP02 mode. Analysis of the theoretical lateral point spread functions for the case of LP01 and LP02 illumination agrees well with the experimental data. As the design space of waveguides and long-period gratings allows for further optimization of the beam parameters of the generated Bessel-like beams in an all-fiber module, this approach offers a robust and yet flexible alternative to free-space optics approaches or the use of conical fiber tips.

Identification of toxic mold species through Raman spectroscopy of fungal conidia.

We use a 785 nm shifted excitation Raman difference (SERDS) technique to measure the Raman spectra of the conidia of 10 mold species of especial toxicological, medical, and industrial importance, including Stachybotrys chartarum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and others. We find that both the pure Raman and fluorescence signals support the hypothesis that for an excitation wavelength of 785 nm the Raman signal originates from the melanin pigments bound within the cell wall of the conidium. In addition, the major features of the pure Raman spectra group into profiles that we hypothesize may be due to differences in the complex melanin biosynthesis pathways. We then combine the Raman spectral data with neural network models to predict species classification with an accuracy above 99%. Finally, the Raman spectral data of all species investigated is made freely available for download and use.

Light and corona: guided-wave readout for coronavirus spike protein-host-receptor binding.

We show that waveguide sensors can enable a quantitative characterization of coronavirus spike glycoprotein-host-receptor binding-the process whereby coronaviruses enter human cells, causing disease. We demonstrate that such sensors can help quantify and eventually understand kinetic and thermodynamic properties of viruses that control their affinity to targeted cells, which is known to significantly vary in the course of virus evolution, e.g., from SARS-CoV to SARS-CoV-2, making the development of virus-specific drugs and vaccine difficult. With the binding rate constants and thermodynamic parameters as suggested by the latest SARS-CoV-2 research, optical sensors of SARS-CoV-2 spike protein-receptor binding may be within sight.

Laser spectroscopic technique for direct identification of a single virus I: FASTER CARS.

From the famous 1918 H1N1 influenza to the present COVID-19 pandemic, the need for improved viral detection techniques is all too apparent. The aim of the present paper is to show that identification of individual virus particles in clinical sample materials quickly and reliably is near at hand. First of all, our team has developed techniques for identification of virions based on a modular atomic force microscopy (AFM). Furthermore, femtosecond adaptive spectroscopic techniques with enhanced resolution via coherent anti-Stokes Raman scattering (FASTER CARS) using tip-enhanced techniques markedly improves the sensitivity [M. O. Scully, et al, Proc. Natl. Acad. Sci. U.S.A. 99, 10994-11001 (2002)].

Multi-pulse laser-induced bubble formation and nanoparticle aggregation using MoS2 nanoparticles.

Understanding of how particles and light interact in a liquid environment is vital for optical and biological applications. MoS2 has been shown to enhance nonlinear optical phenomena due to the presence of a direct excitonic resonance. Its use in biological applications is predicated on knowledge of how MoS2 interacts with ultrafast (< 1 ps) pulses. In this experiment, the interaction between two femtosecond pulses and MoS2 nanoparticles suspended in liquid is studied. We found that the laser pulses induce bubble formation on the surface of a nanoparticle and a nanoparticle aggregate then forms on the surface of the trapped bubble. The processes of formation of the bubble and the nanoparticle aggregation are intertwined.

Insect flight velocity measurement with a CW near-IR Scheimpflug lidar system.

Flight velocity measurement is an important aspect of insect research that can aid insect identification and facilitate studies and monitoring of insect movements. We propose a novel scheme for the 1-D flight velocity measurement of insects, based on a near-IR Scheimpflug lidar system. We implement this new technique and apply it to study insects at the Salter Research Farm, Robertson County, Texas. The resolution property perpendicular to the probing direction of the Scheimpflug lidar system is explored and reveals the capability of retrieving the velocity component normal to the probing direction of insects passing through the field of view of our system. We observe a shift in wingbeat frequency, which indicates the presence of new insect species during the multi-day measurement. The study on 1-D flight velocity reveals a net directional movement of insects, providing supportive evidence of the arrival of a new species.

Adaptive optics approach to surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) spectroscopy is a popular technique for detecting chemicals in small quantities. Rough metallic surfaces with nanofeatures are some of the most widespread and commercially successful substrates for efficient SERS measurements. A rough metallic surface creates a high-density random distribution of so-called "hot spots" with local optical field enhancement causing Raman signal to increase. In this Letter, we revisit the classic SERS experiment [Surf. Sci.158, 229 (1985)SUSCAS0039-602810.1016/0039-6028(85)90297-3] with rough metallic surfaces covered by a thin layer of copper phthalocyanine molecules. As a modification to the classic configuration, we apply an adaptive wavefront correction of a laser beam profile. As a result, we demonstrate an increase in brightness of local SERS hot spots and redistribution of Raman signal over the substrate area. We hypothesize that the improvement is due to optimal coupling of the shaped laser beam to the random plasmonic nanoantenna configurations. We show that the proposed adaptive-SERS modification is independent of the exact structure of the surface roughness and topography, works with many rough surfaces, and gives brighter Raman hot spots in comparison with conventional SERS measurements. We prove that the adaptive SERS is a powerful instrument for improving SERS sensitivity.

Gap-Mode Tip-Enhanced Raman Scattering on Au Nanoplates of Varied Thickness.

Gold nanoplates (AuNPLs) enable the gap-mode configuration of tip-enhanced Raman spectroscopy (TERS). This allows for low-concentration molecular sensing and high-resolution imaging. Compared with non-gap-mode TERS, the gap plasmon provides significantly higher enhancement factors. In addition, AuNPLs exhibit a lightning rod or edge effect, further enhancing the laser field and increasing the spectroscopic sensitivity. In this study, we investigate the relationship between the thickness of AuNPLs and the intensity of the spontaneous Raman signal produced by 4-nitrobenzenethiol, a reporter molecule used in TERS. Our experimental and theoretical results show that the intensity of TERS spectra increases with an increase in the thickness of the AuNPLs. This study of the thickness dependence of AuNPL allows us to find a configuration with maximal nanoplasmonic effects. Moreover, the electromagnetic interaction of the AuNPL with the tip, positioned near the AuNPL's edge, results in a plasmonic nanoantenna configuration for field enhancement, with important promise for future applications to nanobioimaging and biosensing.