Brillouin microscopy monitors rapid responses in subcellular compartments.

Measurements and imaging of the mechanical response of biological cells are critical for understanding the mechanisms of many diseases, and for fundamental studies of energy, signal and force transduction. The recent emergence of Brillouin microscopy as a powerful non-contact, label-free way to non-invasively and non-destructively assess local viscoelastic properties provides an opportunity to expand the scope of biomechanical research to the sub-cellular level. Brillouin spectroscopy has recently been validated through static measurements of cell viscoelastic properties, however, fast (sub-second) measurements of sub-cellular cytomechanical changes have yet to be reported. In this report, we utilize a custom multimodal spectroscopy system to monitor for the very first time the rapid viscoelastic response of cells and subcellular structures to a short-duration electrical impulse. The cytomechanical response of three subcellular structures - cytoplasm, nucleoplasm, and nucleoli - were monitored, showing distinct mechanical changes despite an identical stimulus. Through this pioneering transformative study, we demonstrate the capability of Brillouin spectroscopy to measure rapid, real-time biomechanical changes within distinct subcellular compartments. Our results support the promising future of Brillouin spectroscopy within the broad scope of cellular biomechanics.

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.

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.

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.

Ghost translation: an end-to-end ghost imaging approach based on the transformer network.

Artificial intelligence has recently been widely used in computational imaging. The deep neural network (DNN) improves the signal-to-noise ratio of the retrieved images, whose quality is otherwise corrupted due to the low sampling ratio or noisy environments. This work proposes a new computational imaging scheme based on the sequence transduction mechanism with the transformer network. The simulation database assists the network in achieving signal translation ability. The experimental single-pixel detector's signal will be 'translated' into a 2D image in an end-to-end manner. High-quality images with no background noise can be retrieved at a sampling ratio as low as 2%. The illumination patterns can be either well-designed speckle patterns for sub-Nyquist imaging or random speckle patterns. Moreover, our method is robust to noise interference. This translation mechanism opens a new direction for DNN-assisted ghost imaging and can be used in various computational imaging scenarios.

Entangled photons enabled time-frequency-resolved coherent Raman spectroscopy and applications to electronic coherences at femtosecond scale.

Quantum entanglement has emerged as a great resource for spectroscopy and its importance in two-photon spectrum and microscopy has been demonstrated. Current studies focus on the two-photon absorption, whereas the Raman spectroscopy with quantum entanglement still remains elusive, with outstanding issues of temporal and spectral resolutions. Here we study the new capabilities provided by entangled photons in coherent Raman spectroscopy. An ultrafast frequency-resolved Raman spectroscopy with entangled photons is developed for condensed-phase molecules, to probe the electronic and vibrational coherences. Using quantum correlation between the photons, the signal shows the capability of both temporal and spectral resolutions not accessible by either classical pulses or the fields without entanglement. We develop a microscopic theory for this Raman spectroscopy, revealing the electronic coherence dynamics even at timescale of 50fs. This suggests new paradigms of optical signals and spectroscopy, with potential to push detection below standard quantum limit.

Synthesizing Five-Body Interaction in a Superconducting Quantum Circuit.

Synthesizing many-body interaction Hamiltonians is a central task in quantum simulation. However, it is challenging to synthesize Hamiltonians that have more than two spins in a single term. Here we synthesize m-body spin-exchange Hamiltonians with m up to 5 in a superconducting quantum circuit by simultaneously exciting multiple independent qubits with time-energy correlated photons generated from a qudit. The dynamic evolution of the m-body interaction is governed by the Rabi oscillation between two m-spin states, in which the states of each spin are different. We demonstrate the scalability of our approach by comparing the influence of noises on the three-, four- and five-body interaction and building a many-body Mach-Zehnder interferometer which potentially has a Heisenberg-limit sensitivity. This study paves a way for quantum simulation involving many-body interaction Hamiltonians such as lattice gauge theories in quantum circuits.

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.

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.

Mammalian complex III heme dynamics studied with pump-probe spectroscopy and red light illuminations.

The electronic or molecular mechanisms that initiate photobiomodulation (PBM) in cells are not yet fully understood. The porcine complex III (C-III) of the electron transport chain was characterized with transient absorption spectroscopy (TAS). We then applied our recently developed continuous wave laser coupled TAS procedure (CW-TAS) to investigate the effect of red light irradiances on the heme dynamics of C-III in its c1 reduced state. The time constants were found to be 3.3 ± 0.3 ps for vibrational cooling of the oxidized state and 4.9 ± 0.4 ps for rebinding of the photodissociated axial ligand of the c1 reduced state. The analysis of the CW-TAS procedure yielded no significant changes in the C-III heme dynamics. We rule out the possibility of 635 nm CW light at 4.7 mW/cm2 inducing a PBM effect on the heme dynamic of C-III, specifically with the photodissociation of its axial ligand.

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.

Transient absorption spectroscopy to explore cellular pathways to photobiomodulation.

Photobiomodulation (PBM) describes the use of low irradiance light in the red to near-infrared wavelength range to stimulate biological effects in tissue, and many biological and spectroscopic techniques are used to study PBM. However, these techniques focus on the products or downstream effects rather than the electronic transitions that initiate the PBM processes. This study presents a novel approach to studying low irradiance light exposures on individual proteins and/or protein complexes by combining a continuous wave (CW) laser diode with femtosecond transient absorption spectroscopy (TAS), coined here as CW-TAS, and tests the system on reduced cytochrome c (Cyt c) for proof of principle. TAS was conducted using a 532-nm excitation pump beam and a 350-600 nm supercontinuum probe. CW laser diodes with wavelengths of 450 nm, 635 nm, and 808 nm were interchangeably fiber coupled into the HELIOS Fire. Samples of Cyt c were tested by TAS using a pump power of 15 µW, both with and without CW exposure. CW exposures were carried out with irradiances of 1.60 and 3.20 mW/cm2, except for 808 nm, which was only tested at 1.60 mW/cm2. Both kinetic and global analyses were performed on the TAS data and the time constants for sets with and without CW exposures were compared. The TAS data for Cyt c with the full dosage of CW exposures did not alter the TAS data distinguishably from the control data. No new electronic transient signals were observed beyond the background when testing Cyt c with the CW exposures. Kinetic analysis confirmed that existing transients did not deviate beyond uncertainty. Global time constants for Cyt c were calculated to be 0.25 ± 0.03 ps and 5.1 ± 0.3 ps for the control study, and the time constants for the CW exposed Cyt c were not significantly different. This study concludes that CW irradiation, at doses delivered, does not alter the transient absorption data of Cyt c. The CW-TAS method provides a new tool for studying PBM effects in other proteins and protein complexes that might respond to the CW wavelengths, such as Complex IV, in future studies.

Raman Spectroscopy as a Robust New Tool for Rapid and Accurate Evaluation of Drought Tolerance Levels in Both Genetically Diverse and Near-Isogenic Maize Lines.

Improving drought tolerance of crops has become crucial due to the current scenario of rapid climate change. In particular, development of new maize germplasm with increased drought tolerance is viewed as a major breeding goal to ensure sustainable food and feed production. Therefore, accurate rapid phenotyping techniques for selection of superior maize genotypes are required. The objectives of this study were to determine whether Raman microscopy technique can be applied for accurate assessment of drought-tolerance levels in both genetically diverse and near-isogenic maize lines that differ in their levels of drought-tolerance. Carotenoid degradation is known to be a direct stress response initiated by reactive oxygen species during osmotic stress such as drought. Using Raman mapping, we observed real-time changes in the rate of carotenoid degradation in chloroplasts that was dependent on the strength of osmotic stress. In addition, we showed that the rate of carotenoid degradation as measured by Raman spectroscopy correlates directly with drought tolerance levels of diverse maize genotypes. We conclude that Raman technique is a robust, biochemically selective and non-invasive phenotyping technique that accurately distinguishes drought tolerance levels in both genetically diverse and near-isogenic maize genotypes. We conclude that this technique can be further developed to render it suitable for field-based early assessment of breeding materials with superior drought-tolerance traits.

Sub-Rayleigh second-order correlation imaging using spatially distributive colored noise speckle patterns.

We present a novel method, to our knowledge, to synthesize non-trivial speckle patterns that can enable sub-Rayleigh second-order correlation imaging. The speckle patterns acquire a unique anti-correlation in the spatial intensity fluctuation by introducing the blue noise distribution on spatial Fourier power spectrum to the input light fields through amplitude modulation. Illuminating objects with the blue noise speckle patterns can lead to a sub-diffraction limit imaging system with a resolution more than three times higher than first-order imaging, which is comparable to the resolving power of ninth order correlation imaging with thermal light. Our method opens a new route towards non-trivial speckle pattern generation by tailoring amplitudes in spatial Fourier power spectrum of the input light fields and provides a versatile scheme for constructing sub-Rayleigh imaging and microscopy systems without invoking complicated higher-order correlations.

Unruh and Cherenkov Radiation from a Negative Frequency Perspective.

A ground-state atom uniformly accelerated through the Minkowski vacuum can become excited by emitting an Unruh-Minkowski photon. We show that from the perspective of an accelerated atom, the sign of the frequency of the Unruh-Minkowski photons can be positive or negative depending on the acceleration direction. The accelerated atom becomes excited by emitting an Unruh-Minkowski photon which has negative frequency in the atom's frame, and decays by emitting a positive-frequency photon. This leads to interesting effects. For example, the photon emitted by accelerated ground-state atom cannot be absorbed by another ground-state atom accelerating in the same direction, but it can be absorbed by an excited atom or a ground-state atom accelerated in the opposite direction. We also show that similar effects take place for Cherenkov radiation. Namely, a Cherenkov photon emitted by an atom cannot be absorbed by another ground-state atom moving with the same velocity, but can be absorbed by an excited atom or a ground-state atom moving in the opposite direction.