Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Advancing the study of protein-G4 interactions in DNA repair: Insights from biolayer interferometry.

Biolayer interferometry (BLI) is a powerful tool that enables direct observations of protein-G4 interactions in real-time. In this article, we discuss the crucial aspects in conducting a BLI experiment by using the TAR DNA-binding protein (TDP43) and a G4 DNA formed by (GGGGCC)4 as a sample application. We also describe the necessary precautions in designing the DNA substrate and evaluating the signal contributions arising from nonspecific binding interactions. A comprehensive guide is included that details the necessary materials and reagents, experimental procedures, and data analysis methods for researchers who are interested in using BLI for similar studies. The insights provided in this article will allow researchers to harness the potential of BLI and unravel the complexities of protein-G4 interactions with precision and confidence.

Analyzer-free hard x-ray interferometry.

Objective. To enable practical interferometry-based phase contrast CT using standard incoherent x-ray sources, we propose an imaging system where the analyzer grating is replaced by a high-resolution detector. Since there is no need to perform multiple exposures (with the analyzer grating at different positions) at each scan angle, this scheme is compatible with continuous-rotation CT apparatus, and has the potential to reduce patient radiation dose and patient motion artifacts.Approach. Grating-based x-ray interferometry is a well-studied technique for imaging soft tissues and highly scattering objects embedded in such tissues. In addition to the traditional x-ray absorption-based image, this technique allows reconstruction of the object phase and small-angle scattering information. When using conventional incoherent, polychromatic, hard x-ray tubes as sources, three gratings are usually employed. To sufficiently resolve the pattern generated in these interferometers with contemporary x-ray detectors, an analyzer grating is used, and consequently multiple images need to be acquired for each view angle. This adds complexity to the imaging system, slows image acquisition and thus increases sensitivity to patient motion, and is not dose efficient. By simulating image formation based on wave propagation, and proposing a novel phase retrieval algorithm based on a virtual grating, we assess the potential of a analyzer-grating-free system to overcome these limitations.Main results. We demonstrate that the removal of the analyzer-grating can produce equal image contrast-to-noise ratio at reduced dose (by a factor of 5), without prolonging scan duration.Significance.By demonstrating that an analyzer-free CT system, in conjuction with an efficient phase retrieval algorithm, can overcome the prohibitive dose and workflow penalties associated grating-stepping, an alternative path towards realizing clinical inteferometric CT appears possible.

Tapered Fiber Bioprobe Based on U-Shaped Fiber Transmission for Immunoassay.

In this paper, a tapered fiber bioprobe based on Mach-Zehnder interference (MZI) is proposed. To retain the highly sensitive straight-tapered fiber MZI sensing structure, we designed a U-shaped transmission fiber structure for the collection of optical sensing signals to achieve a miniature-insert-probe design. The spectrum responses from the conventional straight-tapered fiber MZI sensor and our proposed sensor were compared and analyzed, and experimental results showed that our proposed sensor not only has the same sensing capability as the straight-tapered fiber sensor, but also has the advantages of being flexible, convenient, and less liquid-consuming, which are attributed to the inserted probe design. The tapered fiber bioprobe obtained a sensitivity of 1611.27 nm/RIU in the refractive index detection range of 1.3326-1.3414. Finally, immunoassays for different concentrations of human immunoglobulin G were achieved with the tapered fiber bioprobe through surface functionalization, and the detection limit was 45 ng/mL. Our tapered fiber bioprobe has the insert-probe advantages of simpleness, convenience, and fast operation. Simultaneously, it is low-cost, highly sensitive, and has a low detection limit, which means it has potential applications in immunoassays and early medical diagnosis.

High energy x-ray Talbot-Lau interferometer employing a microarray anode-structured target source to extend the field of view.

Objective. High energy and large field of view (FOV) phase contrast imaging is crucial for biological and even medical applications. Although some works have devoted to achieving a large FOV at high energy through bending gratings and so on, which would be extremely challenging in medical high energy imaging.Approach.We analyze the angular shadowing effect of planar gratings in high-energy x-ray Talbot-Lau interferometer (XTLI). Then we design and develop an inverse XTLI coupled with a microarray anode-structured target source to extend the FOV at high energy.Main results.Our experimental results demonstrate the benefit of the source in the inverse XTLI and a large FOV of 106.6 mm in the horizontal direction is achieved at 40 keV. Based on this system, experiments of a mouse demonstrate the potential advantage of phase contrast mode in imaging lung tissue.Significance.We extend the FOV in a compact XTLI using a microarray anode-structured target source coupled with an inverse geometry, which eliminates grating G0 and relaxes the fabrication difficulty of G2. We believe the established design idea and imaging system would facilitate the wide applications of XTLI in high energy phase contrast imaging.

Comparison of the ocular ultrasonic and optical biometry devices in the different quality measurements.

PURPOSE: To compare the reliability and agreement of axial length (AL), anterior chamber depth (ACD), and lens thickness (LT) measurements obtained with optical biometry based on swept-source optical coherence tomography (IOLMaster 700; Carl Zeiss, Germany) and an ultrasound biometry device (Nidek; US-4000 Echoscan, Japan) in different qualities of AL measurement. METHODS: A total of 239 consecutive eyes of 239 cataract surgery candidates with a mean age of 56 ± 14 years were included. The quality measurements were grouped according to the quartiles of SD of the measured AL by IOLMaster 700. The first and fourth quartile's SD are defined as high and low-quality measurement, respectively, and the second and third quartiles' SD is defined as moderate-quality. RESULTS: The reliability of AL and ACD between the two devices in all patients and in different quality measurement groups was excellent with highly statistically significant (AL: all ICC=0.999 and P<0.001, ACD: all ICC>0.920 and P<0.001). AL and ACD in all quality measurements showed a very strong correlation between devices with highly statistically significant. However, there was poor (ICC=0.305), moderate (ICC=0.742), and good (ICC=0.843) reliability in measuring LT in low-, moderate-, and high-quality measurements, respectively. LT showed a very strong correlation (r = 0.854) with highly statistically significant (P<0.001) between devices only in patients with high-quality measurements. CONCLUSIONS: AL and ACD of the IOLMaster700 had outstanding agreements with the US-4000 ultrasound in different quality measurements of AL and can be used interchangeably. But LT should be used interchangeably cautiously only in the high-quality measurements group.

Atom Interferometric Imaging of Differential Potentials Using an Atom Laser.

Interferometry is a prime technique for modern precision measurements. Atoms, unlike light, have significant interactions with electric, magnetic, and gravitational fields, making their use in interferometric applications particularly versatile. Here, we demonstrate atom interferometry to image optical and magnetic potential landscapes over an area exceeding 240 µm×600 µm. The differential potentials employed in our experiments generate phase imprints in an atom laser that are made visible through a Ramsey pulse sequence. We further demonstrate how advanced pulse sequences can enhance desired imaging features, e.g., to image steep potential gradients. A theoretical discussion is presented that provides a semiclassical analysis and matching numerics.

Modified Mach-Zehnder interferometer for spatial coherence measurement.

Spatial coherence of light sources is usually obtained by using the classical Young's interferometer. Although the original experiment was improved upon in successive works, some drawbacks still remain. For example, several pairs of points must be used to obtain the complex coherence degree (normalized first-order correlation function) of the source. In this work, a modified Mach-Zehnder interferometer which includes a pair of lenses and is able to measure the spatial coherence degree is presented. With this modified Mach-Zehnder interferometer, it is possible to measure the full 4D spatial coherence function by displacing the incoming beam laterally. To test it, we have measured only a 2D projection (zero shear) of the 4D spatial coherence, which is enough to characterize some types of sources. The setup has no movable parts, making it robust and portable. To test it, the two-dimensional spatial coherence of a high-speed laser with two cavities was measured for different pulse energy values. We observe from the experimental measurements that the complex degree of coherence changes with the selected output energy. Both laser cavities seem to have similar complex coherence degrees for the maximum energy, although it is not symmetrical. Thus, this analysis will allow us to determine the best configuration of the double-cavity laser for interferometric applications. Furthermore, the proposed approach can be applied to any other light sources.

Towards virtual histology with X-ray grating interferometry.

Breast cancer is the most common type of cancer worldwide. Diagnosing breast cancer relies on clinical examination, imaging and biopsy. A core-needle biopsy enables a morphological and biochemical characterization of the cancer and is considered the gold standard for breast cancer diagnosis. A histopathological examination uses high-resolution microscopes with outstanding contrast in the 2D plane, but the spatial resolution in the third, Z-direction, is reduced. In the present paper, we propose two high-resolution table-top systems for phase-contrast X-ray tomography of soft-tissue samples. The first system implements a classical Talbot-Lau interferometer and allows to perform ex-vivo imaging of human breast samples with a voxel size of 5.57 µm. The second system with a comparable voxel size relies on a Sigray MAAST X-ray source with structured anode. For the first time, we demonstrate the applicability of the latter to perform X-ray imaging of human breast specimens with ductal carcinoma in-situ. We assessed image quality of both setups and compared it to histology. We showed that both setups made it possible to target internal features of breast specimens with better resolution and contrast than previously achieved, demonstrating that grating-based phase-contrast X-ray CT could be a complementary tool for clinical histopathology.

Epitope Binning of Monoclonal and Polyclonal Antibodies by Biolayer Interferometry.

Understanding the epitopes of antibodies elicited by infection and vaccination is often useful in immunogen design. In this chapter, we describe biolayer interferometry (BLI)-based methods to evaluate such epitopes and permit simultaneous analysis of antibodies from several sources, including monoclonal antibodies (mAbs) and polyclonal serum antibodies (pAbs). Using previously characterized antibodies with known epitopes as controls, the distribution of epitopes for the influenza hemagglutinin (HA) is shown for isolated human mAbs and pooled serum from HA-immunized mice. This method is versatile, high-throughput, and can be adapted to several antigens.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

Spectrally Selective Time-Resolved Emission through Fourier-Filtering (STEF).

We demonstrate a method for separating and resolving the dynamics of multiple emitters without the use of conventional filters. By directing the photon emission through a fixed path-length imbalanced Mach-Zehnder interferometer, we interferometrically cancel (or enhance) certain spectral signatures corresponding to one emissive species. Our approach, Spectrally selective Time-resolved Emission through Fourier-filtering (STEF), leverages the detection and subtraction of both outputs of a tuned Mach-Zehnder interferometer, which can be combined with time-correlated single photon counting (TCSPC) or confocal imaging to demix multiple emitter signatures. We develop a procedure to calibrate out imperfections in Mach-Zehnder interferometry schemes. Additionally, we demonstrate the range and utility of STEF by performing the following procedures with one measurement: (1) filtering out laser scatter from a sample, (2) separating and measuring a fluorescence lifetime from a binary chromophore mixture with overlapped emission spectra, (3) confocally imaging and separately resolving the standard fluorescent stains in bovine pulmonary endothelial cells and nearly overlapping fluorescent stains on RAW 264.7 cells. This form of spectral balancing can allow for robust and tunable signal sorting.

Technical Design Considerations of a Human-Scale Talbot-Lau Interferometer for Dark-Field CT.

Computed tomography (CT) as an important clinical diagnostics method can profit from extension with dark-field imaging, as it is currently restricted to X-rays' attenuation contrast only. Dark-field imaging allows access to more tissue properties, such as micro-structural texture or porosity. The up-scaling process to clinical scale is complex because several design constraints must be considered. The two most important ones are that the finest grating is limited by current manufacturing technology to a [Formula: see text] period and that the interferometer should fit into the CT gantry with minimal modifications only. In this work we discuss why an inverse interferometer and a triangular G1 profile are advantageous and make a compact and sensitive interferometer implementation feasible. Our evaluation of the triangular grating profile reveals a deviation in the interference pattern compared to standard grating profiles, which must be considered in the subsequent data processing. An analysis of the grating orientation demonstrates that currently only a vertical layout can be combined with cylindrical bending of the gratings. We also provide an in-depth discussion, including a new simulation approach, of the impact of the extended X-ray source spot which can lead to large performance loss and present supporting experimental results. This analysis reveals a vastly increased sensitivity to geometry and grating period deviations, which must be considered early in the system design process.

Modeling Vibrations of a Tiled Talbot-Lau Interferometer on a Clinical CT.

X-ray computed tomography (CT) is an invaluable imaging technique for non-invasive medical diagnosis. However, for soft tissue in the human body the difference in attenuation is inherently small. Grating-based X-ray phase-contrast is a relatively novel imaging method which detects additional interaction mechanisms between photons and matter, namely refraction and small-angle scattering, to generate additional images with different contrast. The experimental setup involves a Talbot-Lau interferometer whose susceptibility to mechanical vibrations hindered acquisition schemes suitable for clinical routine in the past. We present a processing pipeline to identify spatially and temporally variable fluctuations occurring in an interferometer installed on a continuously rotating clinical CT gantry. The correlations of the vibrations in the modular grating setup are exploited to identify a small number of relevant fluctuation modes, allowing for a sample reconstruction free of vibration artifacts.

Ultrasensitive deafness gene DNA hybridization detection employing a fiber optic Mach-Zehnder interferometer: Enabled by a black phosphorus nanointerface.

The rapid and efficient detection of deafness gene DNA plays an important role in the clinical diagnosis of deafness diseases. This study demonstrates the ultrasensitive detection of complementary DNA (cDNA) by employing a nanointerface-sensitized fiber optic biosensor. The sensor consists of SMF-TNCF-MMF-SMF (abbreviated as STMS) structure with lateral offset. Besides, it is functionalized with a nanointerface of black phosphorus (BP) to enhance the light-matter interaction and eventually improve the sensing performances. Relying on this nanointerface-sensitized sensor, we successfully realize the in-situ detection of cDNA at concentrations ranging from 1 pM to 1 µM, with a sensitivity of 0.719 nm/lgM. The limit of detection (LOD) is as low as 0.24 pM, which is at least two orders of magnitude lower than those of existing methods. The sensor exhibits the advantages of simple operation, fast response, label-free measurement, excellent repeatability, and high selectivity. Our contribution suggests a convenient approach for deafness gene DNA detection and can be extended for general ultra-low concentration DNA detection applications.

Coaxial interferometry for camera-based ultrasound-modulated optical tomography with paired illumination.

Ultrasound-modulated optical tomography (UOT), which combines the advantages of both light and ultrasound, is a promising imaging modality for deep-tissue high-resolution imaging. Among existing implementations, camera-based UOT gains huge advances in modulation depth through parallel detection. However, limited by the long exposure time and the slow framerate of modern cameras, the measurement of UOT signals always requires holographic methods with additional reference beams. This requirement increases system complexity and is susceptible to environmental disturbances. To overcome this challenge, we develop coaxial interferometry for camera-based UOT in this work. Such a coaxial scheme is enabled by employing paired illumination with slightly different optical frequencies. To measure the UOT signal, the conventional phase-stepping method in holography can be directly transplanted into coaxial interferometry. Specifically, we performed both numerical investigations and experimental validations for camera-based UOT under the proposed coaxial scheme. One-dimensional imaging for an absorptive target buried inside a scattering medium was demonstrated. With coaxial interferometry, this work presents an effective way to reduce system complexity and cope with environmental disturbances for camera-based UOT.

Fabrication of Beta-Barium Borate Sensing Head for Non-Invasive Measurement of Fluidic Concentration Variations.

In this study, a beta-barium borate sensing head (BBO-SH) was fabricated and evaluated for the measurements of fluidic concentration variations by using a non-invasive technique. The BBO-SH could be coupled to a fluidic container through thin interlayer water in a heterodyne interferometer based on the phase interrogation. To ensure the sensing head's stability, the package of BBO-SH uses the prism and the coverslip bounded with UV glue, which can resist environmental damage due to moisture. After each use, the sensing head could be easily cleaned. The sensitivity of the BBO-SH remained stable after repeated measurements over a period of 139 days. Finally, the achievable measurement resolutions of the concentration and refractive index are 52 ppm and 1 × 10-6 RIU, respectively, for the sodium chloride solution. The achievable measurement resolutions of the concentration and refractive index were 55 ppm and 8.8 × 10-7 RIU, respectively, for the hydrochloric acid solution.

Long Period Grating Mach-Zehnder Interferometer Based Immunosensor with Temperature and Bulk Refractive Index Compensation.

A long period grating Mach-Zehnder interferometer (LPGMZI) that consists of two identical long period gratings (LPGs) in a single fibre was developed to measure immunoglobulin M (IgM). The measured spectrum has fringes due to the interference between the core mode and cladding mode. This immunosensor inherits the advantages of an LPG and has the potential to compensate for unwanted signal changes due to bulk refractive index (RI) and temperature fluctuations by analysing interference fringes and their envelope. The external RI was measured from 1.3384 to 1.3670 in two different cases: (i) only the connecting section between the two LPGs is immersed or (ii) the whole LPGMZI is immersed. The fringes shift with an external RI in both scenarios, whereas the envelope stays still in case (i) or shifts at the same rate as the fringes in case (ii). The LPGMZI was also characterised at different temperatures between 25 °C and 30 °C by placing the whole LPGMZI in a water bath. The fringes and envelope shift at the same rate with temperature. The LPGMZI platform was then used to create an IgM immunosensor. The connecting section between the two LPGs was functionalised with anti-IgM and immersed into solutions with IgM concentrations from 20 µg/mL to 320 µg/mL. The fringes shift with IgM concentration and the envelope remains static. The results from this work show that LPGMZI has the potential to compensate for the temperature and bulk RI fluctuations and perform as a portable biosensor platform.

Application of attention-DnCNN for ESPI fringe patterns denoising.

Fringe patterns' denoising in electronic speckle pattern interferometry (ESPI) is an important step in phase extraction. In this study, we propose a new denoising method for ESPI fringe patterns based on a convolutional neural network (CNN). The proposed model which combines the attention mechanism and CNN is defined as attention-denoising CNN. In this model, owing to the attention mechanism, more attention will be paid to fringe information, and better filtering results will be achieved. The experimental results show that our proposed method can obtain excellent results, especially with high and large variation density ESPI fringe patterns.

Protein conformation characterization via a silicon resonator-based optical sensor based on the combination of wavelength interrogation and dual polarization detection.

Protein conformational abnormality causes cell malfunction. Conformational change of amyloid protein causes neuron malfunction, which renders "protein conformational disease" Alzheimer's disease. Dual polarization interferometry enables to provide one-dimensional structure of a protein biolayer via deconvolution of interference patterns, which in turn is interpreted as the protein molecule conformation. However, it is still challenging to avoid interference patterns becoming faint and obscure sometimes. Resonance wavelength response to the biolayer structure can achieve a very low detection limit due to inherent high Q factor of an optical resonator. Here, we introduce the concept of combining dual polarization detection with wavelength interrogation via a simple and compact resonator-based optical biosensor. Biolayer were probed by the wave of dual polarization and its opto-geometrical parameters were resolved into resonance wavelength shift. Because protein molecule with distinct conformation produced a biolayer with unique thickness and mass density. Amyloid proteins in monomeric and dimeric morphology were respectively characterized. This concept enables protein conformation characterization in an easy and direct paradigm and provides a desirable sensing performance due to sensitive resonance response in the form of the sharp resonance profile occurring in a nonoverlapping spectrum.