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.

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.

Electrodeposition of Lithium-Based Upconversion Nanoparticle Thin Films for Efficient Perovskite Solar Cells.

In this work, high-quality lithium-based, LiYF4=Yb3+,Er3+ upconversion (UC) thin film was electrodeposited on fluorene-doped tin oxide (FTO) glass for solar cell applications. A complete perovskite solar cell (PSC) was fabricated on top of the FTO glass coated with UC thin film and named (UC-PSC device). The fabricated UC-PSC device demonstrated a higher power conversion efficiency (PCE) of 19.1%, an additional photocurrent, and a better fill factor (FF) of 76% in comparison to the pristine PSC device (PCE = ~16.57%; FF = 71%). Furthermore, the photovoltaic performance of the UC-PSC device was then tested under concentrated sunlight with a power conversion efficiency (PCE) of 24% without cooling system complexity. The reported results open the door toward efficient PSCs for renewable and green energy applications.

Lightly Boron-Doped Nanodiamonds for Quantum Sensing Applications.

Unlike standard nanodiamonds (NDs), boron-doped nanodiamonds (BNDs) have shown great potential in heating a local environment, such as tumor cells, when excited with NIR lasers (808 nm). This advantage makes BNDs of special interest for hyperthermia and thermoablation therapy. In this study, we demonstrate that the negatively charged color center (NV) in lightly boron-doped nanodiamonds (BNDs) can optically sense small temperature changes when heated with an 800 nm laser even though the correct charge state of the NV is not expected to be as stable in a boron-doped diamond. The reported BNDs can sense temperature changes over the biological temperature range with a sensitivity reaching 250 mK/√Hz. These results suggest that BNDs are promising dual-function bio-probes in hyperthermia or thermoablation therapy as well as other quantum sensing applications, including magnetic sensing.

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.

Dynamic nitrogen vacancy magnetometry by single-shot optical streaking microscopy.

Nitrogen vacancy diamonds have emerged as sensitive solid-state magnetic field sensors capable of producing diffraction limited and sub-diffraction field images. Here, for the first time, to our knowledge, we extend those measurements to high-speed imaging, which can be readily applied to analyze currents and magnetic field dynamics in circuits on a microscopic scale. To overcome detector acquisition rate limitations, we designed an optical streaking nitrogen vacancy microscope to acquire two-dimensional spatiotemporal kymograms. We demonstrate magnetic field wave imaging with micro-scale spatial extent and ~400 µs temporal resolution. In validating this system, we detected magnetic fields down to 10 µT for 40 Hz magnetic fields using single-shot imaging and captured the spatial transit of an electromagnetic needle at streak rates as high as 110 µm/ms. This design has the capability to be readily extended to full 3D video acquisition by utilizing compressed sensing techniques and a potential for further improvement of spatial resolution, acquisition speed, and sensitivity. The device opens opportunities to many potential applications where transient magnetic events can be isolated to a single spatial axis, such as acquiring spatially propagating action potentials for brain imaging and remotely interrogating integrated circuits.

Lithium-Based Upconversion Nanoparticles for High Performance Perovskite Solar Cells.

In this work, we report an easy, efficient method to synthesize high quality lithium-based upconversion nanoparticles (UCNPs) which combine two promising materials (UCNPs and lithium ions) known to enhance the photovoltaic performance of perovskite solar cells (PSCs). Incorporating the synthesized YLiF4:Yb,Er nanoparticles into the mesoporous layer of the PSCs cells, at a certain doping level, demonstrated a higher power conversion efficiency (PCE) of 19%, additional photocurrent, and a better fill factor (FF) of 82% in comparison to undoped PSCs (PCE = ~16.5%; FF = 71%). The reported results open a new avenue toward efficient PSCs for renewable energy applications.

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.

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.

Engineering Red-Enhanced and Biocompatible Upconversion Nanoparticles.

The exceptional optical properties of lanthanide-doped upconversion nanoparticles (UCNPs) make them among the best fluorescent markers for many promising bioapplications. To fully utilize the unique advantages of the UCNPs for bioapplications, recent significant efforts have been put into improving the brightness of small UCNPs crystals by optimizing dopant concentrations and utilizing the addition of inert shells to avoid surface quenching effects. In this work, we engineered bright and small size upconversion nanoparticles in a core-shell-shell (CSS) structure. The emission of the synthesized CSS UCNPs is enhanced in the biological transparency window under biocompatible excitation wavelength by optimizing dopant ion concentrations. We also investigated the biosafety of the synthesized CSS UCNP particles in living cell models to ensure bright and non-toxic fluorescent probes for promising bioapplications.

Fluorescent nanodiamonds for luminescent thermometry in the biological transparency window.

Fluorescent nanodiamonds (FNDs) have attracted recent interest for biological applications owing to their biocompatibility and photostability (absence of photoblinking and bleaching). For optical thermometry, nitrogen-vacancy (NV) color centers and silicon-vacancy color centers in diamonds have demonstrated potential, where the NV has the highest sensitivity. However, NV is often excited with green light, which can cause heating and photodamage to tissues, as well as autofluorescence that decreases sensitivity. To overcome these limitations, we report temperature sensing using NV centers excited by deep red light (660 nm), plus another color center that can be excited with NIR light; the nickel (Ni) complex. The NV center measures temperature using diamond lattice expansion while the Ni complex measures temperature using phonon sideband strength.

Engineering water-tolerant core/shell upconversion nanoparticles for optical temperature sensing.

Luminescence thermometry is a promising approach using upconversion nanoparticles (UCNPs) with a nanoscale regime in biological tissues. UCNPs are superior to conventional fluorescent markers, benefiting from their autofluorescence suppression and deep imaging in tissues. However, they are still limited by poor water solubility and weak upconversion luminescence intensity, especially at a small particle size. Recently, YVO4:Er+3,Yb+3 nanoparticles have shown high efficiency upconversion (UC) luminescence in water at single-particle level and high contrast imaging in biological models. Typically, a 980-nm laser triggers the UC process in the UCNPs, which overlaps with maximum absorption of water molecules that are dominant in biological samples, resulting in biological tissues overheating and possible damaging. Interestingly, neodymium (Nd+3) possesses a large absorption cross section at the water low absorption band (808 nm), which can overcome overheating issues. In this Letter, we introduce Nd+3 as a new near-infrared absorber and UC sensitizer into YVO4:Er+3,Yb+3 nanoparticles in a core/shell structure to ensure successive energy transfer between the new UC sensitizer (Nd+3) to the upconverting activator (Er+3). Finally, we synthesized water-tolerant YVO4:Er+3,Yb+3@Nd+3 core/shell nanoparticles (average size 20 nm) with strong UC luminescence at a biocompatible excitation wavelength for optical temperature sensing where overheating in water is minimized.

High resolution fluorescence bio-imaging upconversion nanoparticles in insects.

Imaging fluorescent markers with brightness, photostability, and continuous emission with auto fluorescence background suppression in biological samples has always been challenging due to limitations of available and economical techniques. Here we report a new approach, to achieve high contrast imaging inside small and difficult biological systems with special geometry such as fire ants, an important agricultural pest, using a homemade cost-effective optical system. Unlike the commonly used rare-earth doped fluoride nanoparticles, we utilized nanoparticles with a high upconversion efficiency in water. Specifically Y2O3:Er+3,Yb+3 nanoparticles (40-50 nm diameter) were fed to fire ants as food and then a simple illuminating experiment was conducted at 980 nm wavelength at relatively low pump intensity8 kW.cm-2. The locations were further confirmed by X-ray tomography, where most particles aggregated inside the ant's mouth. High resolution, fast, and economical optical imaging system opens the door for studying more complex biological systems.

High efficiency upconversion nanophosphors for high-contrast bioimaging.

Upconversion nanoparticles (UCNPs) are of interest because they allow suppression of tissue autofluorescence and are therefore visible deep inside biological tissue. Compared to upconversion dyes, UCNPs have a lower pump intensity threshold, better photostability, and less toxicity. Recently, YVO4: Er+3, Yb+3 nanoparticles were shown to exhibit strong up-conversion luminescence with a relatively low 10 kW cm-2 excitation intensity even in water, which makes them excellent bio-imaging candidates. Herein, we investigate their use as internal probes in insects by injecting YVO4 : Er+3, Yb+3 nanoparticles into fire ants as a biological model, and obtain 2D optical images with 980 nm illumination. High-contrast images with high signal-to-noise ratio are observed by detecting the up-conversion fluorescence as the excitation laser is scanned.

Molecular-sized fluorescent nanodiamonds.

Doping of carbon nanoparticles with impurity atoms is central to their application. However, doping has proven elusive for very small carbon nanoparticles because of their limited availability and a lack of fundamental understanding of impurity stability in such nanostructures. Here, we show that isolated diamond nanoparticles as small as 1.6 nm, comprising only ∼400 carbon atoms, are capable of housing stable photoluminescent colour centres, namely the silicon vacancy (SiV). Surprisingly, fluorescence from SiVs is stable over time, and few or only single colour centres are found per nanocrystal. We also observe size-dependent SiV emission supported by quantum-chemical simulation of SiV energy levels in small nanodiamonds. Our work opens the way to investigating the physics and chemistry of molecular-sized cubic carbon clusters and promises the application of ultrasmall non-perturbative fluorescent nanoparticles as markers in microscopy and sensing.

Electron spin resonance shift and linewidth broadening of nitrogen-vacancy centers in diamond as a function of electron irradiation dose.

A high-nitrogen-concentration diamond sample was subjected to 200-keV electron irradiation using a transmission electron microscope. The optical and spin-resonance properties of the nitrogen-vacancy (NV) color centers were investigated as a function of the irradiation dose up to 6.4 × 10(21) e(-)/cm(2). The microwave transition frequency of the NV(-) center was found to shift by up to 0.6% (17.1 MHz) and the linewidth broadened with increasing electron-irradiation dose. Unexpectedly, the measured magnetic sensitivity is best at the lowest irradiation dose, even though the NV concentration increases monotonically with increasing dose. This is in large part due to a sharp reduction in optically detected spin contrast at higher doses.

Slow light for deep tissue imaging with ultrasound modulation.

Slow light has been extensively studied for applications ranging from optical delay lines to single photon quantum storage. Here, we show that the time delay of slow-light significantly improves the performance of the narrowband spectral filters needed to optically detect ultrasound from deep inside highly scattering tissue. We demonstrate this capability with a 9 cm thick tissue phantom, having 10 cm(-1) reduced scattering coefficient, and achieve an unprecedented background-free signal. Based on the data, we project real time imaging at video rates in even thicker phantoms and possibly deep enough into real tissue for clinical applications like early cancer detection.

In situ metal tip sharpening of scanning probe microscopes.

This article demonstrated a new approach for fabrication and sharpening of metal tips of scanning probe microscopes. Experimentally, a metal tip was heated and melted by a focused laser light. The tip was then sharpened by a strong electric field and consolidated as the laser was turned off. With a low-vacuum and a high-voltage source, a 25-µm indium-coated platinum wire was sharpened to a tip with diameters below 50 nm. The minimal tip radius by this method is estimated to be below 1 nm. With this technique, in situ tip sharpening for SPM would be possible.

Highly efficient FRET from a single nitrogen-vacancy center in nanodiamonds to a single organic molecule.

We show highly efficient fluorescence resonance energy transfer (FRET) between negatively charged nitrogen-vacancy (NV) centers in diamond as donor and dye molecules as acceptor, respectively. The energy transfer efficiency is 86% with particles of 20 nm in size. Calculated and experimentally measured energy transfer efficiencies are in excellent agreement. Owing to the small size of the nanocrystals and careful surface preparation, energy transfer between a single nitrogen-vacancy center and a single quencher was identified by the stepwise change of energy transfer efficiencies due to bleaching of single acceptor molecules. Our studies pave the way toward FRET-based scanning probe techniques using single NV donors.

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.