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.

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.

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)].

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.

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.

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.

Tip-Enhanced Raman Imaging of Single-Stranded DNA with Single Base Resolution.

Tip-enhanced Raman scattering (TERS) is a promising optical and analytical technique for chemical imaging and sensing at single molecule resolution. In particular, TERS signals generated by a gap-mode configuration where a silver tip is coupled with a gold substrate can resolve a single-stranded DNA (ssDNA) molecule with a spatial resolution below 1 nm. To demonstrate the proof of subnanometer resolution, we show direct nucleic acid sequencing using TERS of a phage ssDNA (M13mp18). M13mp18 provides a known sequence and, through our deposition strategy, can be stretched (uncoiled) and attached to the substrate by its phosphate groups, while exposing its nucleobases to the tip. After deposition, we scan the silver tip along the ssDNA and collect TERS signals with a step of 0.5 nm, comparable to the bond length between two adjacent DNA bases. By demonstrating the real-time profiling of a ssDNA configuration and furthermore, with unique TERS signals of monomeric units of other biopolymers, we anticipate that this technique can be extended to the high-resolution imaging of various nanostructures as well as the direct sequencing of other important biopolymers including RNA, polysaccharides, and polypeptides.

Fluorescence imaging of stained red blood cells with simultaneous resonance Raman photostability analysis.

Optical spectroscopic imaging of biological systems has important applications in medical diagnosis, biochemistry, and image-guided surgery. Vibrational spectroscopy, such as Raman scattering, provides high chemical selectivity but is limited by weak signals and a large fluorescence background. Fluorescence imaging is often used by introducing specific dyes in biological systems to label different system parts and to increase the image contrast. However, the extrinsic fluorescence of the staining molecules often masks the intrinsic vibrational signals of biomolecules, which could also be simultaneously detected using the same excitation laser source. Therefore, fluorescence staining is often accompanied by the loss of other important complimentary information. For example, the high laser power often used for the rapid, high-quality imaging could lead to photo-induced suppression or bleaching of the fluorescence and Raman signals resulting in sample photodamage. Therefore, simultaneous imaging and photodamage analysis need to be performed in a controlled bioimaging experiment. Here we perform simultaneous spectroscopic bioimaging and photostability analysis of rhodamine 6G (R6G) stained red blood cells (RBCs) using both fluorescence and resonance Raman imaging in a single 532 nm laser excitation experiment. We develop a corresponding data processing algorithm which allows separation of the two spectroscopic signals. We control the relative intensity of the R6G and RBC signals by varying the excitation laser power and simultaneously monitor the photostability of RBCs. We observe no significant photodamage of RBCs through the absence of changes in the relative Raman peak intensities. Conversely, the R6G molecules show bleaching with the suppression of both the fluorescence and resonance Raman signals. Our approach may be generalized to other types of stained cells with the appropriate selection of fluorescent dyes and excitation sources.

Rapid detection of drought stress in plants using femtosecond laser-induced breakdown spectroscopy.

Drought stress disrupts the balance of macro- and micronutrients and affects the yield of agriculturally and economically significant plants. Rapid detection of stress-induced changes of relative content of elements such as sodium (Na), potassium (K), calcium (Ca) and iron (Fe) in the field may allow farmers and crop growers to counter the effects of plant stress and to increase their crop return. Unfortunately, the analytical methods currently available are time-consuming, expensive and involve elaborate sample preparation such as acid digestion which hinders routine daily monitoring of crop health on a field scale. We report application of an alternative method for rapid detection of drought stress in plants using femtosecond laser-induced breakdown spectroscopy (LIBS). We demonstrate daily monitoring of relative content of Na, K, Ca and Fe in decorative indoor (gardenia) and cultivated outdoor (wheat) plant species under various degrees of drought stress. The observed differences in spectral and temporal responses indicate different mechanisms of drought resistance. We identify spectroscopic markers of drought stress which allow for distinguishing mild environmental and severe drought stress in wheat and may be used for remote field-scale estimation of plant stress resistance and health.

Single-beam heterodyne FAST CARS microscopy.

We demonstrate, for the first time, single-beam heterodyne FAST CARS imaging without data post-processing and with nonresonant background subtraction in a simple setup via the real-time piezo modulation of the probe delay. Our fast signal acquisition scheme does not require a spatial light modulator in the pulse shaper, and is suitable for high-resolution imaging and time-resolved dynamics. In addition, the spectral detection of the back-scattered FAST CARS signal is incorporated into the pulse shaper, allowing for a compact and more efficient design. Such epi-detection capability is demonstrated by imaging Si and MoS2 microstructures.

Topological charge algebra of optical vortices in nonlinear interactions.

We investigate the transfer of orbital angular momentum among multiple beams involved in a coherent Raman interaction. We use a liquid crystal light modulator to shape pump and Stokes beams into optical vortices with various integer values of topological charge, and cross them in a Raman-active crystal to produce multiple Stokes and anti-Stokes sidebands. We measure the resultant vortex charges using a tilted-lens technique. We verify that in every case the generated beams' topological charges obey a simple relationship, resulting from angular momentum conservation for created and annihilated photons, or equivalently, from phase-matching considerations for multiple interacting beams.

Low wavenumber efficient single-beam coherent anti-Stokes Raman scattering using a spectral hole.

We demonstrate an approach to detect low wavenumber vibrational signals based on single-beam coherent anti-Stokes Raman scattering (CARS) with a spectral hole. Using a 4f pulse shaper for both pulse shaping and signal collection, we achieve an enhanced efficiency in collecting back-reflected CARS signals.

Coherence brightened laser source for atmospheric remote sensing.

We have studied coherent emission from ambient air and demonstrated efficient generation of laser-like beams directed both forward and backward with respect to a nanosecond ultraviolet pumping laser beam. The generated optical gain is a result of two-photon photolysis of atmospheric O(2), followed by two-photon excitation of atomic oxygen. We have analyzed the temporal shapes of the emitted pulses and have observed very short duration intensity spikes as well as a large Rabi frequency that corresponds to the emitted field. Our results suggest that the emission process exhibits nonadiabatic atomic coherence, which is similar in nature to Dicke superradiance where atomic coherence is large and can be contrasted with ordinary lasing where atomic coherence is negligible. This atomic coherence in oxygen adds insight to the optical emission physics and holds promise for remote sensing techniques employing nonlinear spectroscopy.

Ultrafast waveform synthesis and characterization using coherent Raman sidebands in a reflection scheme.

Coherent Raman sidebands have the potential to serve as a source of single cycle pulses. We generate these sidebands by crossing two-color femtosecond laser pulses in a Raman-active crystal. We design a reflection scheme using spherical mirrors to combine coherent Raman sidebands. The sidebands and the driving pulses are refocused back to the Raman crystal and the relative spectral phases are retrieved from an interferogram based on the nonlinear Raman interaction. Furthermore, using a deformable mirror to adjust the spectral phases, we demonstrate that our setup is capable of synthesizing ultrafast waveforms using coherent Raman sidebands.

Generation of octave-spanning supercontinuum by Raman-assisted four-wave mixing in single-crystal diamond.

An octave-spanning coherent supercontinuum is generated by non-collinear Raman-assisted four-wave mixing in single-crystal diamond using 7.7 fs laser pulses that have been chirped to about 420 fs in duration. The use of ultrabroad bandwidth pulses as input results in substantial overlap of the generated spectrum of the anti-Stokes sidebands, creating a phase-locked supercontinuum when all the sidebands are combined to overlap in time and space. The overall bandwidth of the generated supercontinuum is sufficient to support its compression to isolated few-to-single cycle attosecond transients. The significant spectral overlap of adjacent anti-Stokes sidebands allows the utilization of straight-forward spectral interferometry to test the relative phase coherence of the anti-Stokes outputs and is demonstrated here for two adjacent pairs of sidebands. The method can subsequently be employed to set the relative phase of the sidebands for pulse compression and for the synthesis of arbitrary field transients.

Coherent broadband light generation with a double-path configuration.

We generate broadband light by focusing two femtosecond pulses into a Raman-active crystal. By reflecting Raman sideband beams together with the two driving beams back to the same crystal (with a slight spatial offset), we generate sidebands covering a broader spectral range, compared to a single pass. In this novel double-path configuration, multiple Raman sideband beams interact with each other since the phase-matching condition is automatically fulfilled. This scheme enables an enhanced cascaded coherent anti-Stokes scattering process and also doubles the interaction length, thus it allows one to use relatively weak energy pump pulses and thereby avoid optical damage.

Standoff spectroscopy via remote generation of a backward-propagating laser beam.

In an earlier publication we demonstrated that by using pairs of pulses of different colors (e.g., red and blue) it is possible to excite a dilute ensemble of molecules such that lasing and/or gain-swept superradiance is realized in a direction toward the observer. This approach is a conceptual step toward spectroscopic probing at a distance, also known as standoff spectroscopy. In the present paper, we propose a related but simpler approach on the basis of the backward-directed lasing in optically excited dominant constituents of plain air, N(2) and O(2). This technique relies on the remote generation of a weakly ionized plasma channel through filamentation of an ultraintense femtosecond laser pulse. Subsequent application of an energetic nanosecond pulse or series of pulses boosts the plasma density in the seed channel via avalanche ionization. Depending on the spectral and temporal content of the driving pulses, a transient population inversion is established in either nitrogen- or oxygen-ionized molecules, thus enabling a transient gain for an optical field propagating toward the observer. This technique results in the generation of a strong, coherent, counterpropagating optical probe pulse. Such a probe, combined with a wavelength-tunable laser signal(s) propagating in the forward direction, provides a tool for various remote-sensing applications. The proposed technique can be enhanced by combining it with the gain-swept excitation approach as well as with beam shaping and adaptive optics techniques.

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.