Multimodal Non-Destructive In Situ Observation of Crystallinity Changes in High-Density Polyethylene Samples with Relation to Optical Parameters during Tensile Deformation.

Many non-destructive optical testing methods are currently used for material research, providing various information about material parameters. At RECENDT, a multimodal experimental setup has been designed that combines terahertz (THz) spectroscopy, optical coherence tomography (OCT), infrared (IR), and Raman spectroscopy with a tensile test stage. This setup aims to gather material information such as crystallinity and optical parameters of high-density polyethylene (HDPE) during a tensile test. The setup compares common IR and Raman spectroscopy and the less common optical methods THz and OCT. Complementarity is achieved through different frequency ranges and measurement approaches, resulting in different measured optical material parameters and depths. During tensile testing, HDPE samples with varying crystallinity were analysed, and the determined optical parameters such as refractive index, birefringence, scattering coefficient of decay, and penetration depth can be correlated with the change in crystallinity. These findings demonstrate that the optical methods and their outcomes can be interconnected. With further optimization of the experimental setup, it would be possible to observe the alignment of fibres in fibre composite panels and the stress distribution of polymers effectively. This opens interesting possibilities for polymer characterization in the future, including quality control during moulding processes and material testing.

Recognition Imaging of Trace E-cadherins on the Bladder Cancer Cells Surface during Epithelial-Mesenchymal Transition by Force-Distance Curve-Based AFM.

Tumor-associated epithelial-mesenchymal transition (EMT) contains a set of transitional cellular states usually judged by the EMT marker expression. E-cadherin is a down-regulated EMT epithelial marker, and the detection of E-cadherin is challenging on cancer cell surfaces in the middle and late stages of EMT. Here, the trace E-cadherins on the living bladder cancer T24 cell surface during EMT were investigated with force-distance curve-based atomic force microscopy. The results confirmed that T24 cells are still in an intermediate state and can be transferred into the mesenchymal phenotype by long-term TGF-ß1 induction. During EMT, E-cadherins on the T24 cell surface gradually decreased and rarely clustered. E-cadherin is not completely missing, even at the end of EMT, but is too sparse to cluster. This work provides us with a visual understanding of the expression and distribution of trace markers during EMT and a deep comprehension of the indispensable significance of E-cadherin in cancer cells.

In-Mould OCT Sensors Combined with Piezo-Actuated Positioning Devices for Compensating for Displacement in Injection Overmoulding of Optoelectronic Parts.

When overmoulding optoelectronic devices with optical elements, precise alignment of the overmoulded part and the mould is of great importance. However, mould-integrated positioning sensors and actuators are not yet available as standard components. As a solution, we present a mould-integrated optical coherence tomography (OCT) device that is combined with a piezo-driven mechatronic actuator, which is capable of performing the necessary displacement correction. Because of the complex geometric structure optoelectronic devices may have, a 3D imaging method was preferable, so OCT was chosen. It is shown that the overall concept leads to sufficient alignment accuracy and, apart from compensating for the in-plane position error, provides valuable additional information about the sample both before and after the injection process. The increased alignment accuracy leads to better energy efficiency, improved overall performance and less scrap parts, and thus even a zero-waste production process might be feasible.

Highly accurate THz-CT including refraction effects.

The principles of algebraic image reconstruction are applied to THz computed tomography (THz-CT) in order to account for refraction within the sample. Using the nominal sample geometry as a priori knowledge, a highly accurate and robust image reconstruction algorithm based on the physics of geometric optics is presented. The validity of the geometric forward model is verified by a numerical simulation of Maxwell's equations. Furthermore, the developed method is experimentally tested using measurements performed with a fast THz-CT system based on a THz time-domain spectrometer in transmission mode. Automated evaluations of the reconstructed sample cross sections showed an accuracy of <150 µm.

Mid-infrared DMD-based spectral-coding spectroscopy with a supercontinuum laser source.

We present a mid-infrared spectroscopic system based on a spectral-coding approach enabled by a modified digital micromirror device (DMD). A supercontinuum source offering a confined mid-infrared laser beam is employed to perform gas measurements with this system. The performance, flexibility, and programmability enabled by the DMD is experimentally demonstrated by gas-cell measurements (CO2, CH4, N2O, NO2 and CO). Full spectra are acquired in 14 ms at 10 nm spectral resolution and in 3.5 ms at 40 nm spectral resolution. Further, we employ the system for stand-off open-path spatially resolved CO2 measurements that fully exploit the laser emission properties - the bright and highly-collimated supercontinuum beam is scanned by a galvo mirror over a retroreflector array at a scalable remote distance. The measurement concept models a passing gas emitter under lab conditions; time and spatially resolved CO2 absorbance gas-plume images in the mid-infrared range are obtained.

Phase-contrast THz-CT for non-destructive testing.

A new approach for image reconstruction in THz computed tomography (THz-CT) is presented. Based on a geometrical optics model containing the THz signal amplitude and phase, a novel algorithm for extracting an average phase from the measured THz signals is derived. Applying the algorithm results in a phase-contrast sinogram, which is further used for image reconstruction. For experimental validation, a fast THz time-domain spectrometer (THz-TDS) in transmission geometry is employed, enabling CT measurements within several minutes. Quantitative evaluation of reconstructed 3D printed plastic profiles reveals the potential of our approach in non-destructive testing of plastic profiles.

Spectral-Coding-Based Compressive Single-Pixel NIR Spectroscopy in the Sub-Millisecond Regime.

In this contribution, we present a high-speed, multiplex, grating spectrometer based on a spectral coding approach that is founded on principles of compressive sensing. The spectrometer employs a single-pixel InGaAs detector to measure the signals encoded by an amplitude spatial light modulator (digital micromirror device, DMD). This approach leads to a speed advantage and multiplex sensitivity advantage atypical for standard dispersive systems. Exploiting the 18.2 kHz pattern rate of the DMD, we demonstrated 4.2 ms acquisition times for full spectra with a bandwidth of 450 nm (5250-4300 cm-1; 1.9-2.33 µm). Due to the programmability of the DMD, spectral regions of interest can be chosen freely, thus reducing acquisition times further, down to the sub-millisecond regime. The adjustable resolving power of the system accessed by means of computer simulations is discussed, quantified for different measurement modes, and verified by comparison with a state-of-the-art Fourier-transform infrared spectrometer. We show measurements of characteristic polymer absorption bands in different operation regimes of the spectrometer. The theoretical multiplex advantage of 8 was experimentally verified by comparison of the noise behavior of the spectral coding approach and a standard line-scan approach.

Terahertz Time-Domain Polarimetry in Reflection for Film Characterization.

Terahertz time-domain spectroscopy is a useful technique to characterize layered samples and thin films. It gives access to their optical properties and thickness. Such measurements are done in transmission, which requires access to the sample from opposite sides. In reality this is not always possible. In such cases, reflection measurements are the only option, but they are more difficult to implement. Here we propose a method to characterize films in reflection geometry using a polarimetric approach based on the identification of Brewster angle and modeling of the measured signal to extract the refractive index and thickness of the sample. The technique is demonstrated experimentally on an unsupported single layer thin film sample. The extracted optical properties and thickness were in good agreement with established transmission terahertz spectroscopy measurements. The new method has the potential to cover a wide range of applications, both for research and industrial purposes.

Broadband near-infrared hyperspectral single pixel imaging for chemical characterization.

We introduce a compressive sensing based approach for single pixel hyperspectral chemical imaging in a broad spectral range in the near-infrared. Fully integrated MEMS based Fabry-Pérot tunable filter spectrometers and a digital micro-mirror device were employed to achieve spectral and spatial resolution, respectively. The available spectral range from 1500 to 2200 nm covers molecular overtone vibrations enabling chemical identification. Hyperspectral images of different adhesives deposited on a textile were recorded revealing their chemical composition. Furthermore, spectrally resolved near-infrared images with compression rates up to 90% are presented. The approach of single pixel imaging illustrates a promising technology for the infrared spectral range superior to conventionally used costly focal plane arrays.

Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy.

The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S 11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Y cell = 185 µS + j285 µS and Y bacteria = 3 µS + j20 µS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance-capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+) -containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.

Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Quantitative sub-surface and non-contact imaging using scanning microwave microscopy.

The capability of scanning microwave microscopy for calibrated sub-surface and non-contact capacitance imaging of silicon (Si) samples is quantitatively studied at broadband frequencies ranging from 1 to 20 GHz. Calibrated capacitance images of flat Si test samples with varying dopant density (10(15)-10(19) atoms cm(-3)) and covered with dielectric thin films of SiO2 (100-400 nm thickness) are measured to demonstrate the sensitivity of scanning microwave microscopy (SMM) for sub-surface imaging. Using standard SMM imaging conditions the dopant areas could still be sensed under a 400 nm thick oxide layer. Non-contact SMM imaging in lift-mode and constant height mode is quantitatively demonstrated on a 50 nm thick SiO2 test pad. The differences between non-contact and contact mode capacitances are studied with respect to the main parameters influencing the imaging contrast, namely the probe tip diameter and the tip-sample distance. Finite element modelling was used to further analyse the influence of the tip radius and the tip-sample distance on the SMM sensitivity. The understanding of how the two key parameters determine the SMM sensitivity and quantitative capacitances represents an important step towards its routine application for non-contact and sub-surface imaging.

Influenza virus binds its host cell using multiple dynamic interactions.

Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609-9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus-cell binding quantitatively at the molecular level.

Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells.

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.

Diffraction-limited hyperspectral mid-infrared single-pixel microscopy.

In this contribution, we demonstrate a wide-field hyperspectral mid-infrared (MIR) microscope based on multidimensional single-pixel imaging (SPI). The microscope employs a high brightness MIR supercontinuum source for broadband (1.55 [Formula: see text]-4.5 [Formula: see text]) sample illumination. Hyperspectral imaging capability is achieved by a single micro-opto-electro-mechanical digital micromirror device (DMD), which provides both spatial and spectral differentiation. For that purpose the operational spectral bandwidth of the DMD was significantly extended into the MIR spectral region. In the presented design, the DMD fulfills two essential tasks. On the one hand, as standard for the SPI approach, the DMD sequentially masks captured scenes enabling diffraction-limited imaging in the tens of millisecond time-regime. On the other hand, the diffraction at the micromirrors leads to dispersion of the projected field and thus allows for wavelength selection without the application of additional dispersive optical elements, such as gratings or prisms. In the experimental part, first of all, the imaging and spectral capabilities of the hyperspectral microscope are characterized. The spatial and spectral resolution is assessed by means of test targets and linear variable filters, respectively. At a wavelength of 4.15 [Formula: see text] a spatial resolution of 4.92 [Formula: see text] is achieved with a native spectral resolution better than 118.1 nm. Further, a post-processing method for drastic enhancement of the spectral resolution is proposed and discussed. The performance of the MIR hyperspectral microsopce is demonstrated for label-free chemical imaging and examination of polymer compounds and red blood cells. The acquisition and reconstruction of Hadamard sampled 64 [Formula: see text] 64 images is achieved in 450 ms and 162 ms, respectively. Thus, combined with an unprecedented intrinsic flexibiliy gained by a tunable field of view and adjustable spatial resolution, the demonstrated design drastically improves the sample throughput in MIR chemical and biomedical imaging.

Revealing the Effect of Photothermal Therapy on Human Breast Cancer Cells: A Combined Study from Mechanical Properties to Membrane HSP70.

Hyperthermia-induced overexpression of heat shock protein 70 (HSP70) leads to the thermoresistance of cancer cells and reduces the efficiency of photothermal therapy (PTT). In contrast, cancer cell-specific membrane-associated HSP70 has been proven to activate antitumor immune responses. The dual effect of HSP70 on cancer cells inspires us that in-depth research of membrane HSP70 (mHSP70) during PTT treatment is essential. In this work, a PTT treatment platform for human breast cancer cells (MCF-7 cells) based on a mPEG-NH2-modified polydopamine (PDA)-coated gold nanorod core-shell structure (GNR@PDA-PEG) is developed. Using the force-distance curve-based atomic force microscopy (FD-based AFM), we gain insight into the PTT-induced changes in the morphology, mechanical properties, and mHSP70 expression and distribution of individual MCF-7 cells with high-resolution at the single-cell level. PTT treatment causes pseudopod contraction of MCF-7 cells and generates a high level of intracellular reactive oxygen species, which severely disrupt the cytoskeleton, leading to a decrease in cellular mechanical properties. The adhesion maps, which are recorded by aptamer A8 functional probes using FD-based AFM, reveal that PTT treatment causes a significant upregulation of mHSP70 expression and it starts to exhibit a partial aggregation distribution on the MCF-7 cell surface. This work not only exemplifies that AFM can be a powerful tool for detecting changes in cancer cells during PTT treatment but also provides a better view for targeting mHSP70 for cancer therapy.

Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

Imaging and quantifying analysis the binding behavior of PD-L1 at molecular resolution by atomic force microscopy.

Immunotherapy has emerged as an effective treatment modality for cancer. The interaction of programmed cell death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) plays a key role in tumor-related immune escape and has become one of the most extensive targets for immunotherapy. Herein, we investigated the interaction of PD-L1 with its antibody and PD-1 using atomic force microscopy-based single molecule force spectroscopy for the first time. It was found that the PD-L1/anti-PD-L1 antibody complex was easier to dissociate than PD-L1/PD-1. The unbinding forces of specific interaction of PD-L1 on T24 cells with its antibody and PD-1 were quantitatively measured and similar to those on substrate. In addition, the location of PD-L1 on T24 cells was mapped at the single-molecule level by force-volume mapping. The force maps revealed that PD-L1 randomly distributed on T24 cells surface. The recognition events on cells obviously increased after INF-γ treatment, which proved that INF-γ up-regulated the expression of PD-L1 on T24 cells. These findings enrich our understanding of the molecular mechanisms by which PD-L1 interacts with its antibody and PD-1. It provides useful information for the physical factors that is needed to be considered in the design of inhibitors for tumor immunology.

Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy.

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k(off), compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.