Evaluation of Meibomian Gland Structure and Function in Patients with Vernal Keratoconjunctivitis Using Non Contact Meibography and Lipid Layer Interferometry.

PURPOSE: This study evaluated the structural and functional impact of vernal keratoconjunctivitis (VKC) on meibomian glands (MGs) using a combination of noncontact meibography and lipid layer interferometry. METHODS: In this observational study 50 patients with moderate persistent or severe VKC and 50 age-matched controls underwent MG imaging and lipid layer thickness (LLT) measurements with Lipiview II. Image J software was used to assess MG loss (meibograde) in both lids. All patients underwent dry eye evaluation comprising tear break-up time (TBUT), ocular surface staining (OSS), Schirmer I scoring, and meiboscoring (expressibility and quality of meibum secreted). RESULTS: Meibograde, OSS score, and meiboscore was higher in cases (2.68 ± 0.96, 0.580 ± 1.07, and 0.56 ± 0.95 respectively) than controls (1.80 ± 0.67, 0.00 ± 0.00, 0.22 ± 0.47 respectively) (p < 0.001, 0.001, 0.025 respectively). LLT and TBUT was lower in cases (54.58 ± 9.43 nm and 4.92 ± 3.09 sec respectively) than controls (70.14 ± 22.50 nm and 12.02 ± 2.73 sec respectively) (both p's = 0.001). Both groups had comparable Schirmer I scores. CONCLUSION: Children with VKC have significant MG dropouts, deterioration in meibum quality and a thinner and less stable tear film. VKC patients are thus prone to a vicious cycle of inflammation attributable both to the allergic component and to deterioration in MG structure and function. Co-management of MG dysfunction warrants as much attention as the allergic component itself.

Impact of Cyclic Error on Absolute Distance Measurement Based on Optical Frequency Combs.

Absolute distance measurements based on optical frequency combs (OFCs) have greatly promoted advances in both science and technology, owing to the high precision, large non-ambiguity range (NAR), and a high update rate. However, cyclic error, which is extremely difficult to eliminate, reduces the linearity of measurement results. In this study, we quantitatively investigated the impact of cyclic error on absolute distance measurement using OFCs based on two types of interferometry: synthetic wavelength interferometry and single-wavelength interferometry. The numerical calculations indicate that selecting a suitable reference path length can minimize the impact of cyclic error when combining the two types of interferometry. Recommendations for selecting an appropriate synthetic wavelength to address the tradeoff between achieving a large NAR and minimizing the risk of failure when combining the two methods are provided. The results of this study are applicable not only in absolute distance measurements but also in other applications based on OFCs, such as surface profile, vibration analysis, etc.

Identification of exfoliated monolayer hexagonal boron nitride films with a digital color camera under white light illumination.

Optical microscopy with white light illumination has been employed when obtaining exfoliated monolayer hexagonal boron nitride (1L hBN) films from a large number of randomly placed films on a substrate. However, real-time observation of 1L hBN using a color camera under white light illumination remains challenging since hBN is transparent in the visible wavelength range. The poor optical constant of 1L hBN films in microphotographs is significantly improved using a Si substrate coated with a SiNxthin-film (SiNx/Si). When observing hBN thin films on SiNx/Si using a color digital camera in an optical microscope under white light illumination, the clarity of the captured color images depends on the thickness of the SiNxfilm (d). For real-time direct observation, thedwas optimized based on quantitative chromatic studies tailored to Bayer filters of a color image sensor. Through image simulation, it was determined that the color difference between 1L hBN and the bare substrate is maximized atd= 59 or 70 nm, which was experimentally verified. The SiNx/Si with optimizeddvalues visualized 1L hBN films without requiring significant contrast enhancement via image processing under white light illumination in real-time. Furthermore, the captured color photographs facilitate the reliable determination of the number of layers in few-layer hBN films using the contrast of the green channel of the images.

Spectroscopic and Interferometric Sum-Frequency Imaging of Strongly Coupled Phonon Polaritons in SiC Metasurfaces.

Phonon polaritons enable waveguiding and localization of infrared light with extreme confinement and low losses. The spatial propagation and spectral resonances of such polaritons are usually probed with complementary techniques such as near-field optical microscopy and far-field reflection spectroscopy. Here, infrared-visible sum-frequency spectro-microscopy is introduced as a tool for spectroscopic imaging of phonon polaritons. The technique simultaneously provides sub-wavelength spatial resolution and highly-resolved spectral resonance information. This is implemented by resonantly exciting polaritons using a tunable infrared laser and wide-field microscopic detection of the upconverted light. The technique is employed to image hybridization and strong coupling of localized and propagating surface phonon polaritons in a metasurface of SiC micropillars. Spectro-microscopy allows to measure the polariton dispersion simultaneously in momentum space by angle-dependent resonance imaging, and in real space by polariton interferometry. Notably, it is possible to directly image how strong coupling affects the spatial localization of polaritons, inaccessible with conventional spectroscopic techniques. The formation of edge states is observed at excitation frequencies where strong coupling prevents polariton propagation into the metasurface. The technique is applicable to the wide range of polaritonic materials with broken inversion symmetry and can be used as a fast and non-perturbative tool to image polariton hybridization and propagation.

Displacement Sensing for Laser Self-Mixing Interferometry by Amplitude Modulation and Integral Reconstruction.

To robustly and adaptively reconstruct displacement, we propose the amplitude modulation integral reconstruction method (AM-IRM) for displacement sensing in a self-mixing interferometry (SMI) system. By algebraically multiplying the SMI signal with a high-frequency sinusoidal carrier, the frequency spectrum of the signal is shifted to that of the carrier. This operation overcomes the issue of frequency blurring in low-frequency signals associated with continuous wavelet transform (CWT), enabling the precise extraction of the Doppler frequency of the SMI signal. Furthermore, the synchrosqueezing wavelet transform (SSWT) is utilized to enhance the frequency resolution of the Doppler signal. Our experimental results demonstrate that the proposed method achieves a displacement reconstruction accuracy of 21.1 nm (0.89%). Additionally, our simulations demonstrated that this method can accurately reconstruct target displacement under the conditions of time-varying optical feedback intensity or a signal-to-noise ratio (SNR) of 0 dB, with a maximum root mean square (RMS) error of 22.2 nm. These results highlight its applicability in real-world environments. This method eliminates the need to manually determine the window length for time-frequency conversion, calculate the parameters of the SMI system, or add additional optical devices, making it easy to implement.

Reconstitution of the alternative pathway of the complement system enables rapid delineation of the mechanism of action of novel inhibitors.

The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step in vitro reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.

Rapid identity testing of antibody-based hot targeted radionuclide therapies by bio-layer interferometry.

Targeted Radionuclide Therapies (TRT) involve the tailored combination of a therapeutic radionuclide and a targeting molecule, as for instance antibodies or fragments thereof. Despite their short shelf-life, these drug products must meet stringent regulatory standards before use. We introduce a novel, efficient method utilizing Bio-Layer Interferometry (BLI) for rapid identity testing of TRT drug products in less than five minutes. This approach not only reduces radioactive waste but also minimizes operator exposure to radiation. This label-free method has been successfully developed and validated for three different TRT products, ensuring compliance with Good Manufacturing Practices (GMP). Furthermore, we outline our strategic approach to the production and testing of custom biosensors for each product, firmly grounded in Quality-by-Design (QbD) principles.

Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

Interaction Studies of Hexameric and Pentameric IgMs with Serum-Derived C1q and Recombinant C1q Mimetics.

The interaction between IgM and C1q represents the first step of the classical pathway of the complement system in higher vertebrates. To identify the significance of particular IgM/C1q interactions, recombinant IgMs were used in both hexameric and pentameric configurations and with two different specificities, along with C1q derived from human serum (sC1q) and two recombinant single-chain variants of the trimeric globular region of C1q. Interaction and complement activation assays were performed using the ELISA format, and bio-layer interferometry measurements to study kinetic behavior. The differences between hexameric and pentameric IgM conformations were only slightly visible in the interaction assay, but significant in the complement activation assay. Hexameric IgM requires a lower concentration of sC1q to activate the complement compared to pentameric IgM, leading to an increased release of C4 compared to pentameric IgM. The recombinant C1q mimetics competed with sC1q in interaction assays and were able to inhibit complement activation. The bio-layer interferometry measurements revealed KD values in the nanomolar range for the IgM/C1q interaction, while the C1q mimetics exhibited rapid on and off binding rates with the IgMs. Our results make C1q mimetics valuable tools for developing recombinant C1q, specifically its variants, for further scientific studies and clinical applications.

Widespread seawater intrusions beneath the grounded ice of Thwaites Glacier, West Antarctica.

Warm water from the Southern Ocean has a dominant impact on the evolution of Antarctic glaciers and in turn on their contribution to sea level rise. Using a continuous time series of daily-repeat satellite synthetic-aperture radar interferometry data from the ICEYE constellation collected in March-June 2023, we document an ice grounding zone, or region of tidally controlled migration of the transition boundary between grounded ice and ice afloat in the ocean, at the main trunk of Thwaites Glacier, West Antarctica, a strong contributor to sea level rise with an ice volume equivalent to a 0.6-m global sea level rise. The ice grounding zone is 6 km wide in the central part of Thwaites with shallow bed slopes, and 2 km wide along its flanks with steep basal slopes. We additionally detect irregular seawater intrusions, 5 to 10 cm in thickness, extending another 6 km upstream, at high tide, in a bed depression located beyond a bedrock ridge that impedes the glacier retreat. Seawater intrusions align well with regions predicted by the GlaDS subglacial water model to host a high-pressure distributed subglacial hydrology system in between lower-pressure subglacial channels. Pressurized seawater intrusions will induce vigorous melt of grounded ice over kilometers, making the glacier more vulnerable to ocean warming, and increasing the projections of ice mass loss. Kilometer-wide, widespread seawater intrusion beneath grounded ice may be the missing link between the rapid, past, and present changes in ice sheet mass and the slower changes replicated by ice sheet models.

High-Throughput Identification of Single Nanoparticles via Electrochemically Assisted High-Resolution Plasmonic Scattering Interferometric Microscopy.

The identification of nanoparticles within heterogeneous mixtures poses significant challenges due to the similarity in physical properties among different nanomaterials. Here, we present electrochemically assisted high-resolution plasmonic scattering interferometric microscopy (HR-PSIM). This technique allows for the high-throughput identification of nanoparticles by accurately measuring the refractive index of individual nanoparticles without interference from background signals. Through elimination of parabolic scattering interference and employing electrochemical modulation, HR-PSIM demonstrates high spatial resolution and stability against background noise, enabling the differentiation of nanoparticles with closely matched refractive indices, such as Au and Ag nanoparticles. The efficacy of this method is demonstrated through its application in real-time, label-free imaging of nanoparticle electrochemical activity, providing a platform for the precise and high-throughput characterization of nanomaterials. The robustness of our approach against electrochemical interference and its high spatial resolution mark a significant advancement in the field of nanomaterial analysis, promising wide-ranging applications in nanoparticle research and beyond.

Grating (Moiré) Microinterferometric Displacement/Strain Sensor with Polarization Phase Shift.

Grating (moiré) interferometry is one of the well-known methods for full-field in-plane displacement and strain measurement. There are many design solutions for grating interferometers, including systems with a microinterferometric waveguide head. This article proposes a modification to the conventional waveguide interferometer head, enabling the implementation of a polarization fringe phase shift for automatic fringe pattern analysis. This article presents both the theoretical considerations associated with the proposed solution and its experimental verification, along with the concept of in-plane displacement/strain sensing using the described head.

An Improved Data Processing Algorithm for Spectrally Resolved Interferometry Using a Femtosecond Laser.

Spectrally resolved interferometry utilizing a femtosecond laser is widely employed for absolute distance measurement. However, deviations in the output time pulse of the conventional algorithm through inverse Fourier transform are inevitable. Herein, an improved data processing algorithm employing a time-shifting parameter is proposed to improve the accuracy of spectrally resolved interferometry. The principle of the proposed time-shifting algorithm is analyzed theoretically after clarifying the deviation source of the conventional algorithm. Simulation and experimental work were conducted to indicate the improvement in the accuracy of the output absolute distance. The results demonstrated that the proposed algorithm could reduce the deviation of output distances towards the reference values, reaching 0.58 µm by half compared to the conventional algorithm. Furthermore, the measurement uncertainty was evaluated using the Guide to the Expression of Uncertainty in Measurement (GUM), resulting in an expanded uncertainty of 0.71 µm with a 95% confidence.

Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

High-resolution cell imaging using white light phase shifting interferometry and iterative phase deconvolution.

An optimization algorithm is presented for the deconvolution of a complex field to improve the resolution and accuracy of quantitative phase imaging (QPI). A high-resolution phase map can be recovered by solving a constrained optimization problem of deconvolution using a complex gradient operator. The method is demonstrated on phase measurements of samples using a white light based phase shifting interferometry (WLPSI) method. The application of the algorithm on real and simulated objects shows a significant resolution and contrast improvement. Experiments performed on Escherichia coli bacterium have revealed its sub-cellular structures that were not visible in the raw WLPSI images obtained using a five phase shifting method. These features can give valuable insights into the structures and functioning of biological cells. The algorithm is simple in implementation and can be incorporated into other QPI modalities .

Detection of bladder cancer cells using quantitative interferometric label-free imaging flow cytometry.

Bladder cancer is one of the most common cancers with a high recurrence rate. Patients undergo mandatory yearly scrutinies, including cystoscopies, which makes bladder cancer highly distressing and costly. Here, we aim to develop a non-invasive, label-free method for the detection of bladder cancer cells in urine samples, which is based on interferometric imaging flow cytometry. Eight urothelial carcinoma and one normal urothelial cell lines, along with red and white blood cells, imaged quantitatively without staining by an interferometric phase microscopy module while flowing in a microfluidic chip, and classified by two machine-learning algorithms, based on deep-learning semantic segmentation convolutional neural network and extreme gradient boosting. Furthermore, urine samples obtained from bladder-cancer patients and healthy volunteers were imaged, and classified by the system. We achieved accuracy and area under the curve (AUC) of 99% and 97% for the cell lines on both machine-learning algorithms. For the real urine samples, the accuracy and AUC were 96% and 96% for the deep-learning algorithm and 95% and 93% for the gradient-boosting algorithm, respectively. By combining label-free interferometric imaging flow cytometry with high-end classification algorithms, we achieved high-performance differentiation between healthy and malignant cells. The proposed technique has the potential to supplant cystoscopy in the bladder cancer surveillance and diagnosis space.

Photon Phase Delay Sensing with Sub-Attosecond Uncertainty.

The application of statistical estimation theory to Hong-Ou-Mandel interferometry led to enticing results in terms of the detection limit for photon reciprocal delay and polarisation measurement. In the following paper, a fully fibre-coupled setup operating in the telecom wavelength region proves to achieve, for the first time, in common-path Hong-Ou-Mandel-based interferometry, a detection limit for photon phase delay at the zeptosecond scale. The experimental results are then framed in a theoretical model by calculating the Cramer-Rao bound (CRB) and, after comparison with the obtained experimental results, it is shown that our setup attains the optimal measurement, nearly saturating CRB.

A High-Finesse Suspended Interferometric Sensor for Macroscopic Quantum Mechanics with Femtometre Sensitivity.

We present an interferometric sensor for investigating macroscopic quantum mechanics on a table-top scale. The sensor consists of a pair of suspended optical cavities with finesse over 350,000 comprising 10 g fused silica mirrors. The interferometer is suspended by a four-stage, light, in-vacuum suspension with three common stages, which allows for us to suppress common-mode motion at low frequency. The seismic noise is further suppressed by an active isolation scheme, which reduces the input motion to the suspension point by up to an order of magnitude starting from 0.7 Hz. In the current room-temperature operation, we achieve a peak sensitivity of 0.5 fm/Hz in the acoustic frequency band, limited by a combination of readout noise and suspension thermal noise. Additional improvements of the readout electronics and suspension parameters will enable us to reach the quantum radiation pressure noise. Such a sensor can eventually be utilized for demonstrating macroscopic entanglement and for testing semi-classical and quantum gravity models.

Measuring the Acoustic Reflex through the Tympanic Membrane.

INTRODUCTION: The acoustic reflex is the active response of the middle ear to loud sounds, altering the mechanical transfer function of the acoustic energy into the inner ear. Our goal was to observe the effect of the acoustic reflex on the tympanic membrane by identifying a significant nonlinear increase in membrane oscillations. METHODS: By using interferometric spectrally encoded endoscopy, we record the membrane oscillations over time in response to a loud, 200-ms-long acoustic stimulus. RESULTS: A gradual reflex activation is measured between approximately 40 and 100 ms, manifested as a linear 42% increase in the umbo oscillation amplitude. CONCLUSION: The measured oscillations correlate well with those expected from a mechanical model of a damped harmonic oscillator, and the results of this work demonstrate the potential of interferometric spectrally encoded endoscopy to observe unique dynamical processes in the tympanic membrane and in the middle ear.