Methylglyoxal alters collagen fibril nanostiffness and surface potential.

Collagen fibrils are fundamental to the mechanical strength and function of biological tissues. However, they are susceptible to changes from non-enzymatic glycation, resulting in the formation of advanced glycation end-products (AGEs) that are not reversible. AGEs accumulate with aging and disease and can adversely impact tissue mechanics and cell-ECM interactions. AGE-crosslinks have been related, on the one hand, to dysregulation of collagen fibril stiffness and damage and, on the other hand, to altered collagen net surface charge as well as impaired cell recognition sites. While prior studies using Kelvin probe force microscopy (KPFM) have shown the effect glycation has on collagen fibril surface potential (i.e., net charge), the combined effect on individual and isolated collagen fibril mechanics, hydration, and surface potential has not been documented. Here, we explore how methylglyoxal (MGO) treatment affects the mechanics and surface potential of individual and isolated collagen fibrils by utilizing atomic force microscopy (AFM) nanoindentation and KPFM. Our results reveal that MGO treatment significantly increases nanostiffness, alters surface potential, and modifies hydration characteristics at the collagen fibril level. These findings underscore the critical impact of AGEs on collagen fibril physicochemical properties, offering insights into pathophysiological mechanical and biochemical alterations with implications for cell mechanotransduction during aging and in diabetes. STATEMENT OF SIGNIFICANCE: Collagen fibrils are susceptible to glycation, the irreversible reaction of amino acids with sugars. Glycation affects the mechanical properties and surface chemistry of collagen fibrils with adverse alterations in biological tissue mechanics and cell-ECM interactions. Current research on glycation, at the level of individual collagen fibrils, is sparse and has focused either on collagen fibril mechanics, with contradicting evidence, or surface potential. Here, we utilized a multimodal approach combining Kelvin probe force (KPFM) and atomic force microscopy (AFM) to examine how methylglyoxal glycation induces structural, mechanical, and surface potential changes on the same individual and isolated collagen fibrils. This approach helps inform structure-function relationships at the level of individual collagen fibrils.

High-speed telescope autofocus for UAV detection and tracking.

This paper presents the analysis, implementation and experimental evaluation of a high-speed automatic focus module for a telescope-based UAV detection and tracking system. An existing optical drone detection system consisting of two telescopes and deep learning-based object detection is supplemented by suitable linear stages and passive focus algorithms to enable fast automatic focus adjustment. Field tests with the proposed system demonstrate that UAVs flying at speeds of up to 24 m/s towards the system are successfully tracked and kept in focus from more than 4500 m down to 150 m. Furthermore, different search functions and contrast measures are evaluated and it is shown that the Tenengrad operator combined with the Hill Climbing search function achieve the best performance for focusing on fast moving small UAVs.

Reconstructing highly divergent wavefronts from sparse measurements.

The paper presents a concept for the sparse measurement and reconstruction of highly divergent wavefronts enabling measurements at high throughputs and beyond the dynamic range of the wavefront sensor. In the proposed concept, a direct measurement of the wavefront is carried out, where a few segments of the wavefront are measured with Shack-Hartmann sensors (SHSs). In total about 1% of the wavefront is measured and used for the reconstruction of the entire wavefront, which makes the concept suitable for applications where low measurement times are needed. A simulation analysis and an experimental validation of the concept are carried out, and results show that a wavefront with a divergence of 62° can be reconstructed with a root-mean-square error of about 200 nm.

Fast modal reconstruction of large plane wavefronts from sparse measurements using Shack-Hartmann sensors.

A concept for the fast measurement and reconstruction of optical wavefronts using Shack-Hartmann sensors (SHSs) is presented. For wavefronts with a diameter at the scale of several tens of millimeters, hundreds of measurements with an SHS may be necessary to cover the wavefront. In the proposed concept, a few SHSs are used to measure about 2% of the entire wavefront, providing sufficient measurement data for its successful reconstruction. The small number of SHSs mounted in parallel makes the concept suitable for time-critical applications. A simulation analysis is performed, and an experimental validation of the concept is presented, demonstrating that the wavefront can be reconstructed with an RMS error of about 100 nm.

Improving the precision of parallel registration by incorporating a priori information.

This paper presents an algorithm for the precise registration of optical wavefronts. A wavefront exceeding the spatial or dynamic measurement range of a wavefront sensor, e.g. a Shack-Hartmann sensor, can be measured in multiple sub-measurements, each providing a segment of the wavefront. Sensor misalignment during the measurements results in the demand for registration algorithms to precisely reconstruct the entire wavefront from the segments. The proposed algorithm registers the segments in parallel and incorporates a priori information about the uncertainty of the sensor misalignment obtaining high-quality registration. A simulative analysis of the algorithm with respect to sensor misalignment and measurement errors is presented together with an application of the algorithm to a measured divergent wavefront. In the scope of the analysis, the algorithm is compared to state-of-the-art registration algorithms, such as the iterative closest point (ICP) algorithm, where an improvement of the registration performance by a factor of 3 is obtained. Results show that the algorithm is able to reconstruct a divergent and a freeform wavefront with an RMS registration error of a few tens of nanometers with a standard deviation of 80 µm and 2.4 mrad.

AC Kelvin Probe Force Microscopy Enables Charge Mapping in Water.

Mapping charged chemical groups at the solid-liquid interface is important in many areas, ranging from colloidal systems to biomolecular interactions. However, classical methods to measure surface charges either lack spatial resolution or─like Kelvin-probe force microscopy (KPFM)─cannot be applied in aqueous solutions because a DC bias voltage is used. Here, we show that using AC Kelvin probe force microscopy (AC-KPFM), in which the DC bias is replaced with an AC voltage of sufficiently high frequency, the surface potential of spatially fixated, charged surface groups can be mapped in aqueous solution. We demonstrate this with micropatterned, functionalized alkanethiol layers which expose ionized amino- and carboxy-groups. These groups are representative of the charged groups of most biomolecules such as proteins. By adjusting the pH of the solution, the charge of the groups was reversibly altered, demonstrating the electrostatic nature of the measured signal. The influence of the electric double layer (EDL) on the measurement is discussed, and we, furthermore, show how charged, micropatterned layers can be used to spatially direct the deposition of nanoparticles of opposite charge.

Instrument for tensile testing of individual collagen fibrils with facile sample coupling and uncoupling.

Collagen is the major structural protein in human bodies constituting about 30% of the entire protein mass. Through a self-assembly process, triple helical collagen molecules assemble into high aspect-ratio fibers of tens to hundreds of nanometer diameter, known as collagen fibrils (CFs). In the last decade, several methods for tensile testing these CFs emerged. However, these methods are either overly time-consuming or offer low data acquisition bandwidth, rendering dynamic investigation of tensile properties impossible. Here, we describe a novel instrument for tensile testing of individual CFs. CFs are furnished with magnetic beads using a custom magnetic tweezer. Subsequently, CFs are lifted by magnetic force, allowing them to be picked-up by a microgripper structure, which is mounted on a cantilever-based interferometric force probe. A piezo-lever actuator is used to apply tensile displacements and to perform tensile tests of tethered CFs, after alignment. Once the mechanical tests are finished, CFs are removed from the microgripper by application of a magnetic field. Our novel instrument enables tensile tests with at least 25-fold increased throughput compared to tensile testing with an atomic force microscope while achieving force resolution (p-p) of 10 nN at a strain resolution better than 0.1%.

Camera-guided real-time laser ranging for multi-UAV distance measurement.

This paper presents the design and implementation of a scalable laser ranger finder (LRF)-based prototype system, which enables distance measurement and precise localization of multiple unmanned aerial vehicles (UAVs) in real-time. The system consists of a telescope and camera as the image acquisition components, supplemented by an LRF and a fast steering mirror (FSM) to obtain the distance measurement. By combining the optical path of the camera and the LRF through a dichroic mirror, the LRF is accurately aligned by the FSM based on the angular position of a UAV within the camera field of view. The implemented prototype successfully demonstrates distance measurements of up to four UAVs with a bandwidth of 14 Hz per object.

Iterative parallel registration of strongly misaligned wavefront segments.

The paper presents an algorithm for the precise registration of multiple wavefront segments containing large misalignment and phase differences. The measurement of a wavefront with huge dynamics or a large aperture size can be carried out in multiple Shack-Hartmann sensor measurements of segments of the wavefront. The registration algorithm is flexible with respect to the shape of the wavefront and can reconstruct a plane as well as divergent wavefronts, making it suitable for freeform wavefronts. The algorithm enables parallel registration of the wavefront segments which is carried out in an iterative manner to compensate for large misalignment errors. A simulative analysis of the proposed algorithm compares its performance to a fast parallel registration (FPR) algorithm and the established iterative closest point (ICP) algorithm. For a sensor misalignment of up to 100 µm and 3 mrad the algorithm registers a plane and a divergent wavefront with a precision that is a factor 4 and 12 better than the registration precision of the FPR and ICP algorithm.

Adaptive Lissajous scanning pattern design by phase modulation.

This paper proposes a phase modulation method for Lissajous scanning systems, which provides adaptive scan pattern design without changing the frame rate or the field of view. Based on a rigorous analysis of Lissajous scanning, phase modulation constrains and a method for pixel calculation are derived. An accurate and simple metric for resolution calculation is proposed based on the area spanned by neighboring pixels and used for scan pattern optimization also considering the scanner dynamics. The methods are implemented using MEMS mirrors for verification of the adaptive pattern shaping, where a 5-fold resolution improvement in a defined region of interest is demonstrated.

Fast, precise, and shape-flexible registration of wavefronts.

A fast and precise algorithm for wavefront reconstruction by the registration of wavefront segments is presented. If the wavefront exceeds the sensor aperture or the dynamic range of the sensor, a Shack-Hartmann sensor can measure only segments of an optical wavefront. The algorithm registers the wavefront segments in parallel, where they are simultaneously transformed to minimize their overlap mismatch for precise reconstruction of the entire wavefront. The original nonlinear optimization problem is approximated by a convex optimization problem that can be solved more efficiently. A simulation-based analysis of the algorithm and a comparison to a previously proposed parallel registration (PR) algorithm as well as to the iterative closest point (ICP) algorithm are presented. It is shown that despite measurement noise, the algorithm can precisely register plane as well as divergent wavefronts with root mean square registration errors smaller than 10 nm. Particularly for the divergent wavefront, this enables a reduction of the registration error by a factor of up to 750 as compared to the established algorithms. Analysis and comparison to the ICP and PR algorithm also show that the computation time of the proposed algorithm can be from one to three orders of magnitude smaller.

Compact scanning confocal chromatic sensor enabling precision 3-D measurements.

This paper proposes a compact and lightweight scanning confocal chromatic sensor (SCCS) for robot-based precision three-dimensional (3-D) surface measurement applications. The integrated system design includes a 2-D fast steering mirror (FSM) to manipulate the optical path of a high precision 1-D confocal chromatic sensor (CCS). A data-driven calibration procedure is used to accurately combine the FSM deflection angles and the correspondingly measured distances to the sample surface in order to obtain a correctly reconstructed 3-D image. Lissajous scan trajectories are applied to enable efficient scans of the sample surface. The SCCS provides 3-D images at frame rates of up to 1 fps and a measurement volume of 0.35×0.25×1.8mm3, as well as the measurement of arbitrary regions of interest. Using a calibration standard including structures with defined sizes, the lateral and axial resolutions are determined to 2.5 µm and 76 nm, respectively.

Robust wavefront segment registration based on a parallel approach.

This paper presents a robust registration algorithm for wavefront reconstruction from multiple partial measurements. Wavefronts exceeding the dynamic range or size of the Shack-Hartmann sensor can be measured as a set of segments. The wavefront is reconstructed by parallel registration of these wavefront segments, enabling compensation for sensor misalignment as well as for phase differences. For registration, a global mismatch metric is minimized by rigid body transformations and propagation of the wavefront segments. Apart from the description of the algorithm, a simulation-based evaluation and comparison to the iterative closest point (ICP) algorithm is performed. It is shown that in the case of a noisy data set, the parallel approach enables reconstruction errors that are a factor of 10 smaller than the result obtained with the ICP algorithm.

High-speed scanning chromatic confocal sensor for 3-D imaging with modeling-free learning control.

This paper presents a scanning system that integrates a chromatic confocal displacement sensor for topography measurement of a surface. To take an advantage of its compactness and reliability, an off-the-shelf chromatic confocal displacement sensor is integrated. Instead of moving the sensor, a galvanometer scanner reflects the optical point to increase the scan speed, and fast and accurate scanning motion is realized by learning without a model. The resulting images are corrected based on a geometric model to compensate for image distortion.

Confocal chromatic sensor with an actively tilted lens for 3D measurement.

Confocal chromatic displacement sensors are versatile and precise sensors for measuring the distance to a single point. In order to obtain a 3D measurement device, this paper presents an integrated scanning sensor design that employs a tilting lens mechanism for manipulating the light path of the sensor. The optical implications of the design are analytically modeled and simulated. An experimental setup is constructed to evaluate the system design and to test its performance on a variety of samples. Results show good agreement with the simulations and modeling; with maximal tip/tilt angles of ±2.5∘, the setup is capable of measuring a volume of 1.7×1.7×1mm3 with a lateral resolution of 24.8 µm and an axial resolution of 3 µm.

Glycation changes molecular organization and charge distribution in type I collagen fibrils.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Optical scanning of laser line sensors for 3D imaging.

In scanning laser triangulation sensors for 3D imaging, the achievable throughput is strongly limited by the moving mass. By realizing an optical scanning approach rather than repositioning the entire sensor, this limitation could be reduced, leading to a reduced measurement time. This work presents sensor system geometries in which only the optical path of a line triangulation sensor is manipulated by a tip-tilt mirror. In the proposed rotational scanning systems, either the illumination path or both the illumination and the reflection path are manipulated. By using ray-tracing simulation, the performance of the scanning systems are optimized and possible disadvantages can be determined up front. Using geometric relations, the surface profile can be reconstructed from the measured sensor data, the mirror position, and the model parameters. Experimental results show that the image quality of the proposed rotational scanning systems is comparable to systems based on classical translational scanning motion.

Evaluation of surface charge shift of collagen fibrils exposed to glutaraldehyde.

Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale "cables" acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the non-enzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.

Atomic force microscopy capable of vibration isolation with low-stiffness Z-axis actuation.

For high-resolution imaging without bulky external vibration isolation, this paper presents an atomic force microscope (AFM) capable of vibration isolation with its internal Z-axis (vertical) actuators moving the AFM probe. Lorentz actuators (voice coil actuators) are used for the Z-axis actuation, and flexures guiding the motion are designed to have a low stiffness between the mover and the base. The low stiffness enables a large Z-axis actuation of more than 700 µm and mechanically isolates the probe from floor vibrations at high frequencies. To reject the residual vibrations, the probe tracks the sample by using a displacement sensor for feedback control. Unlike conventional AFMs, the Z-axis actuation attains a closed-loop control bandwidth that is 35 times higher than the first mechanical resonant frequency. The closed-loop AFM system has robustness against the flexures' nonlinearity and uses the first resonance for better sample tracking. For further improvement, feedforward control with a vibration sensor is combined, and the resulting system rejects 98.4% of vibrations by turning on the controllers. The AFM system is demonstrated by successful AFM imaging in a vibrational environment.

High bandwidth deflection readout for atomic force microscopes.

This contribution presents the systematic design of a high bandwidth deflection readout mechanism for atomic force microscopes. The widely used optical beam deflection method is revised by adding a focusing lens between the cantilever and the quadrant photodetector (QPD). This allows the utilization of QPDs with a small active area resulting in an increased detection bandwidth due to the reduced junction capacitance. Furthermore the additional lens can compensate a cross talk between a compensating z-movement of the cantilever and the deflection readout. Scaling effects are analyzed to get the optimal spot size for the given geometry of the QPD. The laser power is tuned to maximize the signal to noise ratio without limiting the bandwidth by local saturation effects. The systematic approach results in a measured -3 dB detection bandwidth of 64.5 MHz at a deflection noise density of 62fm/√Hz.