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Label-free detection techniques for single particles and molecules play an important role in basic science, disease diagnostics, and nanomaterial investigations. While fluorescence-based methods are tools for single molecule detection and imaging, they are limited by available molecular probes and photoblinking and photobleaching. Photothermal microscopy has emerged as a label-free imaging technique capable of detecting individual nanoabsorbers with high sensitivity. Whispering gallery mode (WGM) microresonators can confine light in a small volume for enhanced light-matter interaction and thus are a promising ultra-sensitive photothermal microscopy platform. Previously, microtoroid optical resonators were combined with photothermal microscopy to detect 250 nm long gold nanorods and 100 nm long polymers. Here, we combine microtoroids with photothermal microscopy to spatially detect single 5 nm diameter quantum dots (QDs) with a signal-to-noise ratio exceeding 10 4 . Photothermal images were generated by point-by-point scanning of the pump laser. Single particle detection was confirmed for 18 nm QDs by high sensitivity fluorescence imaging and for 5 nm QDs via comparison with theory. Our system demonstrates the capability to detect a minimum heat dissipation of 0.75 pW. To achieve this, we integrated our microtoroid based photothermal microscopy setup with a low amplitude modulated pump laser and utilized the proportional-integral-derivative controller output as the photothermal signal source to reduce noise and enhance signal stability. The heat dissipation of these QDs is below that from single dye molecules. We anticipate that our work will have application in a wide variety of fields, including the biological sciences, nanotechnology, materials science, chemistry, and medicine., (© 2024. The Author(s).)

Whispering gallery mode (WGM) microtoroid resonators are one of the most sensitive biochemical sensors in existence, capable of detecting single molecules. The main barrier for translating these devices out of the laboratory is that light is evanescently coupled into these devices though a tapered optical fiber. This hinders translation of these devices as the taper is fragile, suffers from mechanical vibration, and requires precise positioning. Here, we eliminate the need for an optical fiber by coupling light into and out from a toroid via free-space coupling and monitoring the scattered resonant light. A single long working distance objective lens combined with a digital micromirror device (DMD) was used for light injection, scattered light collection, and imaging. We obtain Q-factors as high as 1.6 × 10 8 with this approach. Electromagnetically induced transparency (EIT)-like and Fano resonances were observed in a single cavity due to indirect coupling in free space. This enables improved sensing sensitivity. The large effective coupling area (~10 μm in diameter for numerical aperture = 0.14) removes the need for precise positioning. Sensing performance was verified by combining the system with the frequency locked whispering evanescent resonator (FLOWER) approach to perform temperature sensing experiments. A thermal nonlinear optical effect was examined by tracking the resonance through FLOWER while adjusting the input power. We believe that this work will be a foundation for expanding the implementation of WGM microtoroid resonators to real-world applications., (© 2024. The Author(s).)

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus mutates, finding effective drugs becomes more challenging. In this study, we use ultrasensitive frequency locked microtoroid optical resonators in combination with in silico screening to search for COVID-19 drugs that can stop the virus from attaching to the human angiotensin-converting enzyme 2 (hACE2) receptor in the lungs. We found 29 promising candidates that could block the binding site and selected four of them that were likely to bind very strongly. We tested three of these candidates using frequency locked optical whispering evanescent resonator (FLOWER), a label-free sensing method based on microtoroid resonators. FLOWER has previously been used for sensing single macromolecules. Here we show, for the first time, that FLOWER can provide accurate binding affinities and sense the inhibition effect of small molecule drug candidates without labels, which can be prohibitive in drug discovery. One of the candidates, methotrexate, showed binding to the spike protein 1.8 million times greater than that to the receptor binding domain (RBD) binding to hACE2, making it difficult for the virus to enter cells. We tested methotrexate against different variants of the SARS-CoV-2 virus and found that it is effective against all four of the tested variants. People taking methotrexate for other conditions have also shown protection against the original SARS-CoV-2 virus. Normally, it is assumed that methotrexate inhibits the replication and release of the virus. However, our findings suggest that it may also block the virus from entering cells. These studies additionally demonstrate the possibility of extracting candidate ligands from large databases, followed by direct receptor-ligand binding experiments on the best candidates using microtoroid resonators, thus creating a workflow that enables the rapid discovery of new drug candidates for a variety of applications., Competing Interests: The authors declare the following competing financial interest(s): J.S. owns a financial stake in Femtorays Technologies which develops label-free molecular sensors., (© 2024 American Chemical Society.)

We present the design and feasibility testing of a multimodal co-registered endoscope based on a dual-path optical system integrated with a scanning piezo. This endoscope incorporates three different imaging modalities. A large field of view reflectance imaging system enables visualization of objects several millimeters in front of the endoscope, while optical coherence microscopy and multiphoton microscopy are employed in contact with tissue to further analyze suspicious areas. The optical system allows multiple different imaging modalities by employing a dual optical path. One path features a low numerical aperture and wide field of view to allow reflectance imaging of distant objects. The other path features a high numerical aperture and short working distance to allow microscopy techniques such as optical coherence microscopy and multiphoton microscopy. Images of test targets were obtained with each imaging modality to verify and characterize the imaging capabilities of the endoscope. The reflectance modality was demonstrated with a 561 nm laser to allow high contrast with blood vessels. It achieved a lateral resolution of 24.8 μm at 5 mm and a working distance from 5 mm to 30 mm. Optical coherence microscopy (OCM) was performed with a 1300 nm super-luminescent diode since this wavelength experiences low relative scattering to allow for deeper tissue imaging. Measured OCM lateral and axial resolution was 4.0 μm and 14.2 μm, respectively. Multiphoton microscopy (MPM) was performed with a custom 1400 nm femtosecond fiber laser, a wavelength suitable for exciting multiple exogenous and some endogenous fluorophores, as well as providing information on tissue composition through harmonic generation processes. A 4.0 μm MPM lateral resolution was measured., Competing Interests: Disclosures The authors declare no conflicts of interest.

Sensitive and rapid biosensors are of critical importance for a variety of applications including infectious disease detection and monitoring as well as medical diagnostics and drug discovery. Whispering gallery mode microtoroid optical resonators are among the most sensitive biochemical sensors in existence. When combined with frequency-locking and data-processing techniques, these sensors have been shown to be capable of single-molecule detection in under 30 s. The sensitivity of these sensors is affected by how a concentration of analyte molecules is transported to the surface of the sensors and the average time it takes the molecules to bind at that concentration. Currently, one question in the field is that at these low concentrations, how these microsensors achieve such rapid response times. Here, we reconcile theory and experiment and demonstrate through flow visualization experiments and finite-element simulations that the total analyte arrival and binding time can be on the order of seconds. This fast response time provides an advantage over nanoscale sensors such as nanowires or nanorods. We anticipate that these results can help us to control, with confidence, when and how many molecules bind to these sensors, thus enabling the building of faster and more sensitive sensors.

The nondiscriminating aspartyl-tRNA synthetase (ND-AspRS), found in many archaea and bacteria, covalently attaches aspartic acid to tRNA Asp and tRNA Asn generating a correctly charged Asp-tRNA Asp and an erroneous Asp-tRNA Asn . This relaxed tRNA specificity is governed by interactions between the tRNA and the enzyme. In an effort to assess the contributions of the anticodon-binding domain to tRNA specificity, we constructed two chimeric enzymes, Chimera-D and Chimera-N, by replacing the native anticodon-binding domain in the Helicobacter pylori ND-AspRS with that of a discriminating AspRS (Chimera-D) and an asparaginyl-tRNA synthetase (AsnRS, Chimera-N), both from Escherichia coli. Both chimeric enzymes showed similar secondary structure compared to wild-type (WT) ND-AspRS and maintained the ability to form dimeric complexes in solution. Although less catalytically active than WT, Chimera-D was more discriminating as it aspartylated tRNA Asp over tRNA Asn with a specificity ratio of 7.0 compared to 2.9 for the WT enzyme. In contrast, Chimera-N exhibited low catalytic activity toward tRNA Asp and was unable to aspartylate tRNA Asn . The observed catalytic activities for the two chimeras correlate with their heterologous toxicity when expressed in E. coli. Molecular dynamics simulations show a reduced hydrogen bond network at the interface between the anticodon-binding domain and the catalytic domain in Chimera-N compared to Chimera-D or WT, explaining its lower stability and catalytic activity., (© 2020 Wiley Periodicals, Inc.)

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNA Asp and tRNA Asn . Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS 1-104 ) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Å resolution. The apo form of H. pylori ND-AspRS 1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNA Asp and tRNA Asn . It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F ( 78 GAGL 81 and 83 NPKL 86 ) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNA Asp ( 34 GUC 36 ) and tRNA Asn ( 34 GUU 36 ). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNA(Asp) and tRNA(Asn) with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and β-sheet secondary structure at 20°C and predominantly β-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein., (Copyright © 2013 Elsevier Inc. All rights reserved.)