Array Of Spots Research Articles

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

Multifocal meta-fiber based on the fractional Talbot effect.

Multi-focusing of light is a crucial capability for photonic devices that can be effectively achieved by precisely modulating the phase delay on the incident wavefront. However, integrating functional structures into optical fibers for remote light focusing remains challenging due to the complex device design and limited fabrication approaches. Here, we present the design and fabrication of metalens array on the end-face of a tailored single-mode step-index fiber for focusing light field into closely packed focal spot array. The metalenses are configured based on the fractional Talbot effect and benefit a modular design capability. Light passing through the optical fiber can be focused into different focal planes. With a synergistic 3D laser nanoprinting technique based on two-photon polymerization, high-quality meta-fibers are demonstrated for focusing light parallelly with a uniform numerical aperture (NA) as high as approximately 0.77. This may facilitate various applications such as optical trapping, generation of sophisticated beam profiles, and boosting light coupling efficiencies.

Propagation properties of partially coherent vector beam with multiple off-axis vortex phases

In this paper, we investigate the propagation properties of radially polarized rectangular-symmetric cosine-Gaussian Schell-model (RCGSM) beam with multiple off-axis vortex phases by using Fourier transform and convolution method based on the unified theory of coherence and polarization. The results indicate that the radially polarized RCGSM beam has self-splitting properties and can be split into four identical lobes due to its unique spatial coherence structure. Furthermore, the modulation of multiple off-axis vortex phases can be acted on each lobe. For high coherence, the spot arrays with triangular or square hollow light intensity distribution can be generated in the focal plane by modulating the number of off-axis vortices (<i>N</i><sub>0</sub>) and the beam order, and the corresponding state of polarization on each lobe presents an inverted triangular or oblique square elliptic distribution. However, for small coherence, the modulation effect of multiple off-axis vortex phases disappears and the light intensity of each lobe degenerates into a quasi-Gaussian distribution, whereas its state of polarization keeps invariant, which is independent of the beam order and coherence length. In addition, the beam still has a certain self-healing ability for one of the off-axis vortex phases partially blocked by an obstacle, but it will be destroyed for completely blocking, resulting in a notch on each lobe.

Simultaneous micromachining with beam splitting of ultrashort laser pulses at high average power for the productive fabrication of microchannels

Laser micromachining with ultrashort laser pulses is a promising approach for the generation of small geometries in the micro-meter scale, such as microchannels for passive fluid transport. The ablation of small structures with high average laser power still poses a challenge when high surface quality is required. In this work, beam splitting with a diffractive optical element (DOE) was used to divide the laser beam of a high-power ultrafast laser into an array of 1x15 spots on the workpiece. Fast scanning of up to 20 m/s perpendicular to the orientation of the array using a Galvanometer-scanner allowed for simultaneous micromachining of multiple microchannels with high surface quality on cemented tungsten carbide. The high average laser power of 160 W in combination with the adapted processing strategy enabled the simultaneous and productive fabrication of 15 microchannels with a low surface roughness of Sa < 1.5 µm along the channel wall.

Comparative Methods for Demystifying Spatial Transcriptomics.

Spatial Transcriptomics (ST), coined as the term for parallel RNA-Seq on cell populations ordered spatially on a histological tissue section, has recently become increasingly popular, especially in experiments where microfluidics-based single-cell sequencing fails, such as assays on neurons. ST platforms, like the 10x Visium technology investigated herein, therefore produce in a single experiment simultaneously thousands of RNA readouts, captured by an array of micrometer scale spots under the histological section. Therefore, a central challenge of analyzing ST experiments consists of analyzing the gene expression morphology of all spots to delineate clusters of similar cell mixtures, which are then compared to each other to identify up- or down-regulated marker genes. Moreover, another level of complexity in ST experiments, compared to traditional RNA-Seq, is imposed by staining the tissue section with protein markers of cells or cell components to identify spots providing relevant information afterward. The corresponding microscopy images need to be analyzed in addition to the RNA-Seq read mappings on the reference genome and transcriptome sequences. Focusing on the software suite provided by the Visium platform manufacturer, we break down the ST analysis pipeline into its four essential steps-the image analysis, the read alignment, the gene quantification, and the spot clustering-and compare results obtained when using reads from different subsets of spots and/or when employing alternative genome or transcriptome references. Our comparative analyses demonstrate the impact of spot selection and the choice of genome/transcriptome references on the analysis results when employing the manufacturer's pipeline.

Angle monitor of micromirror array for freeform illumination in lithography systems

Source and mask optimization is a critical technique for further resolution enhancement in immersion lithography systems, wherein the optimal illumination source shape is widely generated by the micromirror array. Accordingly, the accurate achievement of the allocated angles of micromirrors is a key prerequisite for the implementation of arbitrary illumination patterns. In this paper, we propose an angle monitor to ensure the high-precision tilting of thousands of biaxial micromirrors. As one of the critical modules for closed-loop control of the two-dimensional micromirror array, the online monitor feeds the monitored high-precision tilt angle back to the processing system. The angle monitor mainly consists of the spot scanning module and the angle detection module. Among them, an f-θ lens and a Fourier transform lens with satisfied performances are designed and evaluated by CODE V. Furthermore, a galvanometer and the designed f-θ lens are adopted for the generation of the spot array irradiated on the electrostatic actuated mirrors. Meanwhile, the designed Fourier transform lenses are employed to detect the corresponding tilt angles. In addition, the performance of the proposed system is identified by simulations and experiments individually. It is demonstrated that the monitorable biaxial tilt range of the system is (−2.5°, +2.5°) with a repeatability of better than 0.005°. The large-format micromirror array can be monitored completely without optical cross-talk. Through the device, a 16 × 16 micromirror array is monitored, where the initial angle with no bias voltage applied is captured and the voltage–angle relation for individual micromirrors is obtained. In general, the proposed system can be utilized in the illumination system, providing an efficient and reliable method for complex source shape generation.

Printer toner-assisted immobilization of antibodies on PET for genuinely-2D, flexible ELISA spot arrays

The application of laser printing on a transparent poly(ethylene terephthalate) (PET) foil for toner-assisted immobilization of antibodies is employed for the first time in the preparation of two-dimensional arrays for high throughput ELISA. Two protocols for rapid microfabrication of single- and multi-layer immunoassay substrates with spatially controlled wettability are demonstrated, which include essential steps of laser Printing, UV-ozone Irradiation, and ultrasonically assisted toner Delamination (PID). This study demonstrates the potential of laser toner as an effective and easily removable protectant of PET against permanent hydrophilization by UV irradiation. To prove the feasibility and analytical performance of this novel immunosensing platform, an indirect sandwich immunoassay for human C-reactive protein (hCRP) was selected as a model. Dynamic range, covering CRP levels between 10 ng∙mL−1 and 5.62 µg∙mL−1, with a detection limit of 0.656 ng∙mL−1, as well as excellent signal recovery in human serum (97.3 - 104.5%), confirms clinical suitability of the assay in the analysis of real samples. Thanks to the design and fabrication simplicity, use of commonly available materials and up-scalability, presented methodologies can be successfully applied in the production of substrates for high-throughput immunodiagnostics and flexible microfluidics in Lab-on-a-Foil technology.

Accurate piecewise centroid calculation algorithm for wavefront measurement in adaptive optics.

Adaptive optics using direct wavefront sensing (direct AO) is widely used in two-photon microscopy to correct sample-induced aberrations and restore diffraction-limited performance at high speeds. In general, the direct AO method employs a Sharked-Hartman wavefront sensor (SHWS) to directly measure the aberrations through a spot array. However, the signal-to-noise ratio (SNR) of spots in SHWS varies significantly within deep tissues, presenting challenges for accurately locating spot centroids over a large SNR range, particularly under extremely low SNR conditions. To address this issue, we propose a piecewise centroid calculation algorithm called GCP, which integrates three optimal algorithms for accurate spot centroid calculations under high-, medium-, and low-SNR conditions. Simulations and experiments demonstrate that the GCP can accurately measure aberrations over a large SNR range and exhibits robustness under extremely low-SNR conditions. Importantly, GCP improves the AO working depth by 150 µm compared to the conventional algorithm.

Three-dimensional optical multiple trapping using pure amplitude octagonal almost periodic structures.

We demonstrate a novel method for three-dimensional optical multiple trapping using pure amplitude octagonal almost periodic structures (PAOAPSs). We use a Gaussian beam to diffract through these structures and create a three-dimensional array of trapping spots with the aid of an objective lens. Our device is simple, cost-effective, and easy to fabricate, and it has several advantages over conventional methods for trapping multiple particles. By adjusting the rotation of the PAOAPS and the polarization of the beam, we can simultaneously rotate the trapped particles in both axial and orbital directions. We show that our device achieves an ∼19-fold increase in trapping efficiency compared to a recently introduced method based on an amplitude radial grating. Furthermore, our device transfers about 1/70 of the transmitted beam power to each optical trap, which is much more efficient than a spatial light modulator (SLM).

Integration of high-resolution imaging through scattering medium into a disposable micro-endoscope via projection of 2D spots-array

The objective of this research includes integration of high-resolution imaging through scattering medium, such as blood, into a disposable micro-endoscope. A fiber laser integrated into the micro-endoscope as part of its illumination channel, allows to project a tunable array of spots of light onto an object, that is located behind the scattering medium. We have a laser fiber as part of the illumination channel of a disposable micro-endoscope. By using proper optics, we convert the temporal modulation of the laser into spatial distribution. Thus, the result is generation of spatial spots when using a pulsed laser. The detection channel is a holographic recording of the collected back scattered light, that allows extraction of the electrical field. By time integrating the field we obtain the realization of the spatial array of illumination spots formed on top of the inspected object and behind the scattering medium. By changing the temporal modulation of the illumination laser (changing its temporal photonic signals), we can tune the positions of the spots in the illumination array. If the distance between the projected spots is larger than the imaging resolution, then by applying localization microscopy algorithms together with scanning of the position of the spots in the array, will yield a high-resolution reconstruction of the inspected object. We theoretically and experimentally demonstrate the discussed operation principle and show the potential of the proposed concept as a modality in medical endoscopic procedures.

Complex surface cracks detection and identification by laser array spots thermography with PCA-Frangi processing

In this paper, a method for rapid detection and automatic identification of complex surface cracks based on laser array spots thermography with PCA-Frangi processing is developed. With the Laser Array Spots Thermography (LAST), a complex surface crack in a relatively large surface area can be detected rapidly. In addition, a novel approach utilizing the PCA-Frangi algorithm for feature extraction is proposed to enhance the imaging performance of surface cracks with complex morphology. The results show that the proposed method can extract the cracks and eliminate the interference of array spots to crack identification. Furthermore, this method effectively mitigates the impact of thermal inhomogeneity and thermal noise during the heating process. Finally, the crack features are automatically identified. The simulation and experimental results illustrated that the method enables the accurate recognition of complex surface crack characteristics, further enhancing the overall performance of the crack detection with LAST.

Periodic diffractive optical element for high-density and large-scale spot array structured light projection.

Structured light projection has been widely used for depth sensing in computer vision. Diffractive optical elements (DOEs) play a crucial role in generating structured light projected onto objects, and spot array is a common projection pattern. However, the primary metrics of the spot array, including density and field of view, are restricted by the principle of diffraction and its calculation. In this paper, a novel, to the best of our knowledge, method is proposed to achieve high-density periodic spot array on a large scale. Further, periodic DOEs, for the first time, are optimized to increase the density of the spot array without decreasing the periods of the DOE. Simulation and experimental results of high-density and large-scale spot array structured light projection are presented, demonstrating the effectiveness of the proposed method.

Silver nanosphere array substrates fabricated by glancing angle deposition: Morphology, hot spots and SERS properties

The generation and exploitation of hot spots play a crucial role in the fabrication of highly sensitive surface-enhanced Raman scattering (SERS) substrates. In this work, silver nanosphere (AgNS) array substrates with various morphologies of polystyrene (PS) core/Ag shell were fabricated by reactive ion etching and glancing angle deposition technique on PS sphere templates. The electromagnetic field distribution (hot spots) of AgNS array was calculated by the finite difference time domain method. R6G and MB were used as probe molecules to investigate the effect of structure spacing on electric field enhancement. Under 785 nm excitation, an AgNS substrate with a gap of approximately 20 nm exhibited the best SERS enhancement, and the limit of detection reached 5 × 10−8 M for both analytes. For R6G and MB, the relative standard deviations (RSDs) were 2.39 % and 7.22 %, and the enhancement factors (EFs) were 7.25 × 108 and 7.68 × 107, respectively. These results serve as a reference for the development of new highly sensitive SERS sensors.

Method for testing freeform surfaces based on a Shack-Hartmann sensor with plane wavefront scanning and stitching.

Currently, the surface error measurement technology for freeform faces a significant contradiction between measurement accuracy and dynamic range. The study proposes a non-null testing method for measuring freeform surfaces by utilizing a Shack-Hartmann wavefront sensor to emit a small aperture parallel beam and scan along the normal direction at the center of subapertures for stitching (SHPSS). A mathematical model based on ray tracing and the reflection theorem is established to calculate the sampling points on an ideal freeform surface, the reference spot array on CCD, and the corresponding relationship between microlens array and spots. An algorithm is proposed to iteratively calculate the wavefront aberration and gradually approach the actual sampling points using the established model. Theoretical analysis and numerical simulation results indicate that SHPSS can increase the dynamic range and improve the accuracy of wavefront reconstruction. The error analysis of the SHPSS method is carried out, the measurement accuracy of full aperture freeform surface is 11.45 nm. A testing system is set up and experiments are conducted on a 100 mm aperture freeform reflective mirror. The RMS of the SHPSS test results is less than λ/30 (λ=635 nm) compared to the interferometric test results. By analyzing five groups of repeated measurement experiments, the repeatability accuracy of SHPSS method is less than 1/80 λ (RMS). This demonstrates the feasibility and measurement capabilities of the method for freeform surface testing.

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

Drilling Sequence Optimization Using Evolutionary Algorithms to Reduce Heat Accumulation for Femtosecond Laser Drilling with Multi-Spot Beam Profiles.

We report on laser drilling borehole arrays using ultrashort pulsed lasers with a particular focus on reducing the inadvertent heat accumulation across the workpiece by optimizing the drilling sequence. For the optimization, evolutionary algorithms are used and their results are verified by thermal simulation using Comsol and experimentally evaluated using a thermal imaging camera. To enhance process efficiency in terms of boreholes drilled per second, multi-spot approaches are employed using a spatial light modulator. However, as higher temperatures occur across the workpiece when using simultaneous multi-spot drilling as compared to a single-spot process, a subtle spatial distribution and sequence of the multi-spot approach has to be selected in order to limit the resulting local heat input over the processing time. Different optimization approaches based on evolutionary algorithms aid to select those drilling sequences which allow for the combination of a high efficiency of multi-spot profiles, a low-generated process temperature and a high-component quality. In particular, using a 4 × 4 laser spot array allows for the drilling of 40,000 boreholes in less than 76 s (526 boreholes/s) with a reduced temperature increase by about 35%, as compared to a single spot process when employing an optimized drilling sequence.

Simple Method of Light Field Calculation for Shaping of 3D Light Curves

We propose a method for generating three-dimensional light fields with given intensity and phase distributions using purely phase transmission functions. The method is based on a generalization of the well-known approach to the design of diffractive optical elements that focus an incident laser beam into an array of light spots in space. To calculate purely phase transmission functions, we use amplitude encoding, which made it possible to implement the designed elements using a single spatial light modulator. The generation of light beams in the form of rings, spirals, Lissajous figures, and multi-petal “rose” distributions uniformly elongated along the optical axis in the required segment is demonstrated. It is also possible to control the three-dimensional structure of the intensity and phase of the shaped light fields along the propagation axis. The experimentally generated intensity distributions are in good agreement with the numerically obtained results and show high potential for the application of the proposed method in laser manipulation with nano- and microparticles, as well as in laser material processing.

Rapid reconstruction algorithm for multifocal structured illumination microscopy

Multifocal structured illumination microscopy (MSIM) could surpass the diffraction limit by illuminating the sample with multiple-spot grid patterns. Developed as a parallel version of image scanning microscopy (ISM) to enhance data acquisition speed, MSIM can also be viewed as a variation of structured illumination microscopy (SIM) in which the conventional sinusoidal illumination is substituted with a lattice array of spots. This method outperforms regular SIM when imaging samples that are thick or highly scattered. In this study, we present a rapid reconstruction algorithm for MSIM in the spatial domain and demonstrate that the radius of its equivalent optical transfer function (OTF) support is twice that of a general wide-field microscopy in the epi-illumination geometry. Numerical simulations and experimental results have validated the effectiveness of the proposed algorithm.

Easy to go and innovative validation process using the spot weld inspection system PHAsis and related software

In the industry 4.0 the need for a cost-saving and efficient production process is of high significance. This includes every area of the production processes and leads to appropriate testing procedures by means of the quality management. A combination of ultrasonic inspection devices and software tools for result post processing reduces the need of several cost-intensive validation circles with destructive testing to a very low level. This can be shown for example with the imaging phased array spot weld inspection system PHAsis in combination with a software app for post processing. This is including the software part for the data and result management as well as a device software for the ultrasonic inspection of the spot welds. In combination with the usage of an automation interface for robotic usage, even more cost reduction is possible. Several validation processes on typical resistance spot welding applications with plate pairings of two or three plates (aluminium as well as steel combinations) by the use of PHAsis and the connected PostProcessor will be exemplified. The general procedure of the validation process will be accentuated step by step. Therefore, the original data of phased array testing and a comparative method in the shape of a destructive testing (hammer &amp; chisel test or horizontal respectively vertical cross section) will be presented. Furthermore, the steps of the post processing procedure to define the ultrasound system parameters for a best fit will be described. During these steps, the influence of the different sensitivity and system parameters on the spot weld size will be explained. Also, the measurement uncertainties given by the destructive testing that have an influence to the comparation with ultrasonic testing in general will be addressed. The efficiency of the post processing software tool due to reaching a high match between PHAsis and the comparative destructive method will be illustrated.

Towards scanning nanostructure X-ray microscopy.

This article demonstrates spatial mapping of the local and nanoscale structure of thin film objects using spatially resolved pair distribution function (PDF) analysis of synchrotron X-ray diffraction data. This is exemplified in a lab-on-chip combinatorial array of sample spots containing catalytically interesting nanoparticles deposited from liquid precursors using an ink-jet liquid-handling system. A software implementation is presented of the whole protocol, including an approach for automated data acquisition and analysis using the atomic PDF method. The protocol software can handle semi-automated data reduction, normalization and modeling, with user-defined recipes generating a comprehensive collection of metadata and analysis results. By slicing the collection using included functions, it is possible to build images of different contrast features chosen by the user, giving insights into different aspects of the local structure.