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.

Novel Nanocomposites for Luminescent Thermometry with Two Different Modalities.

In this work, we successfully integrated fluorescent nanodiamonds (FNDs) and lanthanide ion-doped upconversion nanoparticles (UCNPs) in a nanocomposite structure for simultaneous optical temperature sensing. The effective integration of FND and UCNP shells was confirmed by employing high-resolution TEM imaging, X-ray diffraction, and dual-excitation optical spectroscopy. Furthermore, the synthesized ND@UCNP nanocomposites were tested by making simultaneous optical temperature measurements, and the detected temperatures showed excellent agreement within their sensitivity limit. The simultaneous measurement of temperature using two different modalities having different sensing physics but with the same composite nanoparticles inside is expected to greatly improve the confidence of nanoscale temperature measurements. This should resolve some of the controversy surrounding nanoscale temperature measurements in biological applications.

In vivo diagnostics of early abiotic plant stress response via Raman spectroscopy.

Development of a phenotyping platform capable of noninvasive biochemical sensing could offer researchers, breeders, and producers a tool for precise response detection. In particular, the ability to measure plant stress in vivo responses is becoming increasingly important. In this work, a Raman spectroscopic technique is developed for high-throughput stress phenotyping of plants. We show the early (within 48 h) in vivo detection of plant stress responses. Coleus (Plectranthus scutellarioides) plants were subjected to four common abiotic stress conditions individually: high soil salinity, drought, chilling exposure, and light saturation. Plants were examined poststress induction in vivo, and changes in the concentration levels of the reactive oxygen-scavenging pigments were observed by Raman microscopic and remote spectroscopic systems. The molecular concentration changes were further validated by commonly accepted chemical extraction (destructive) methods. Raman spectroscopy also allows simultaneous interrogation of various pigments in plants. For example, we found a unique negative correlation in concentration levels of anthocyanins and carotenoids, which clearly indicates that plant stress response is fine-tuned to protect against stress-induced damages. This precision spectroscopic technique holds promise for the future development of high-throughput screening for plant phenotyping and the quantification of biologically or commercially relevant molecules, such as antioxidants and pigments.

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.

Fluorescent nanodiamond-bacteriophage conjugates maintain host specificity.

Rapid identification of specific bacterial strains within clinical, environmental, and food samples can facilitate the prevention and treatment of disease. Fluorescent nanodiamonds (FNDs) are being developed as biomarkers in biology and medicine, due to their excellent imaging properties, ability to accept surface modifications, and lack of toxicity. Bacteriophages, the viruses of bacteria, can have exquisite specificity for certain hosts. We propose to exploit the properties of FNDs and phages to develop phages conjugated with FNDs as long-lived fluorescent diagnostic reagents. In this study, we develop a simple procedure to create such fluorescent probes by functionalizing the FNDs and phages with streptavidin and biotin, respectively. We find that the FND-phage conjugates retain the favorable characteristics of the individual components and can discern their proper host within a mixture. This technology may be further explored using different phage/bacteria systems, different FND color centers and alternate chemical labeling schemes for additional means of bacterial identification and new single-cell/virus studies.

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.

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.

Nanometer-scale luminescent thermometry in bovine embryos.

Luminescent nanothermometry is a powerful tool that can precisely monitor temperature changes in animal embryos. Among the most sensitive nanoluminescent temperature sensors are fluorescent nanodiamonds (FNDs), having nitrogen-vacancy color centers, and lanthanide-ion-doped upconversion nanoparticles (UCNPs). Here, we investigate their use as nanothermometers inside bovine embryos. The motivation for using both FNDs and UCNPs to measure temperature is to avoid the question of sensor confusion by the local cellular environment. Specifically, by simultaneously measuring temperature using two different modalities having different physics, it is possible to greatly improve the measurement confidence, thereby directly addressing the recent controversy surrounding temperature measurements in living organisms.

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.

All-optical initialization, readout, and coherent preparation of single silicon-vacancy spins in diamond.

The silicon-vacancy (SiV-) color center in diamond has attracted attention because of its unique optical properties. It exhibits spectral stability and indistinguishability that facilitate efficient generation of photons capable of demonstrating quantum interference. Here we show optical initialization and readout of electronic spin in a single SiV- center with a spin relaxation time of T1=2.4±0.2 ms. Coherent population trapping (CPT) is used to demonstrate coherent preparation of dark superposition states with a spin coherence time of T2⋆=35±3 ns. This is fundamentally limited by orbital relaxation, and an understanding of this process opens the way to extend coherence by engineering interactions with phonons. Hyperfine structure is observed in CPT measurements with the 29Si isotope which allows access to nuclear spin. These results establish the SiV- center as a solid-state spin-photon interface.

Nanoscale imaging magnetometry with diamond spins under ambient conditions.

Magnetic resonance imaging and optical microscopy are key technologies in the life sciences. For microbiological studies, especially of the inner workings of single cells, optical microscopy is normally used because it easily achieves resolution close to the optical wavelength. But in conventional microscopy, diffraction limits the resolution to about half the wavelength. Recently, it was shown that this limit can be partly overcome by nonlinear imaging techniques, but there is still a barrier to reaching the molecular scale. In contrast, in magnetic resonance imaging the spatial resolution is not determined by diffraction; rather, it is limited by magnetic field sensitivity, and so can in principle go well below the optical wavelength. The sensitivity of magnetic resonance imaging has recently been improved enough to image single cells, and magnetic resonance force microscopy has succeeded in detecting single electrons and small nuclear spin ensembles. However, this technique currently requires cryogenic temperatures, which limit most potential biological applications. Alternatively, single-electron spin states can be detected optically, even at room temperature in some systems. Here we show how magneto-optical spin detection can be used to determine the location of a spin associated with a single nitrogen-vacancy centre in diamond with nanometre resolution under ambient conditions. By placing these nitrogen-vacancy spins in functionalized diamond nanocrystals, biologically specific magnetofluorescent spin markers can be produced. Significantly, we show that this nanometre-scale resolution can be achieved without any probes located closer than typical cell dimensions. Furthermore, we demonstrate the use of a single diamond spin as a scanning probe magnetometer to map nanoscale magnetic field variations. The potential impact of single-spin imaging at room temperature is far-reaching. It could lead to the capability to probe biologically relevant spins in living cells.

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.

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.

Remote sub-diffraction imaging with femtosecond laser filaments.

Achieving super-resolution has become a scientific imperative for remote imaging of objects and scenes needing increased detail and has motivated the development of various laser-based techniques. We demonstrate a scheme which achieves subdiffraction imaging of remote objects by using femtosecond laser filaments. The use of laser filaments for imaging is destined to have applications in many environments.

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.

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.

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.