Extended-Aperture Shape Measurements Using Spatially Partially Coherent Illumination (ExASPICE).

We have recently demonstrated that the 3D shape of micro-parts can be measured using LED illumination based on speckle contrast evaluation in the recently developed SPICE profilometry (shape measurements based on imaging with spatially partially coherent illumination). The main advantage of SPICE is its improved robustness and measurement speed compared to confocal or white light interferometry. The limited spatial coherence of the LED illumination is used for depth discrimination. An electrically tunable lens in a 4f-configuration is used for fast depth scanning without mechanically moving parts. The approach is efficient, takes less than a second to capture required images, is eye-safe and offers a depth of focus of a few millimeters. However, SPICE's main limitation is its assumption of a small illumination aperture. Such a small illumination aperture affects the axial scan resolution, which dominates the measurement uncertainty. In this paper, we propose a novel method to overcome the aperture angle limitation of SPICE by illuminating the object from different directions with several independent LED sources. This approach reduces the full width at half maximum of the contrast envelope to one-eighth, resulting in a twofold improvement in measurement accuracy. As a proof of concept, shape measurements of various metal objects are presented.

Flash-profilometry: fullfield lensless acquisition of spectral holograms for coherence scanning profilometry.

Flash-profilometry is a novel measurement approach based on the fullfield lensless acquisition of spectral holograms. It is based on spectral sampling of the mutual coherence function and the subsequent calculation of its propagation along the optical axis several times the depth-of-field. Numerical propagation of the entire coherence function, rather than solely the complex amplitude, allows to digitally reproduce a complete scanning white-light interferometric (WLI) measurement. Hence, the corresponding 3D surface profiling system presented here achieves precision in the low nanometer range along an axial measurement range of several hundred micrometers. Due to the lensless setup, it is compact, immune against dispersion effects and lightweight. Additionally, because of the spectral sampling approach, it is faster than conventional coherence scanning WLI and robust against mechanical distortions, such as vibrations and rigid body movements. Flash-profilometry is therefore suitable for a wide range of applications, such as surface metrology, optical inspection, and material science and appears to be particularly suitable for a direct integration into production environments.

Radiolabeling of Platelets with 99mTc-HYNIC-Duramycin for In Vivo Imaging Studies.

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.

Preclinical Characterization of the 177Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Functional pixels: a pathway towards true holographic displays using today's display technology.

Today's 3D dynamic holographic display techniques suffer from severe limitations due to an available number of pixels that is several orders of magnitude lower than required by conventional approaches. We introduce a solution to this problem by introducing the concept of functional pixels. This concept is based on pixels that individually spatially modulate the amplitude and phase of incident light with a polynomial function, rather than just a constant phase or amplitude. We show that even in the simple case of a linear modulation of the phase, the pixel count can be drastically reduced up to 3 orders of magnitude while preserving most of the image details. This scheme can be easily implemented with already existing technology, such as micro mirror arrays that provide tip, tilt and piston movement. Even though the individual pixels need to be technologically more advanced, the comparably small number of such pixels required to form a display may pave the way towards true holographic dynamic 3D displays.

Extending vision ray calibration by determination of focus distances.

The application of cameras as sensors in optical metrology techniques for three-dimensional topography measurement, such as fringe projection profilometry and deflectometry, presumes knowledge regarding the metric relationship between image space and object space. This relation is established by camera calibration and a variety of techniques are available. Vision ray calibration achieves highly precise camera calibration by employing a display as calibration target, enabling the use of active patterns in the form of series of phase-shifted sinusoidal fringes. Besides the required spatial coding of the display surface, this procedure yields additional full-field contrast information. Exploiting the relation between full-field contrast and defocus, we present an extension of vision ray calibration providing the additional information of the focus distances of the calibrated camera. In our experiments we achieve a reproducibility of the focus distances in the order of mm. Using a modified Laplacian based focus determination method, we confirm our focus distance results within a few mm.

Terahertz referenceless wavefront sensing by means of computational shear-interferometry.

In this contribution, we demonstrate the first referenceless measurement of a THz wavefront by means of shear-interferometry. The technique makes use of a transmissive Ronchi phase grating to generate the shear. We fabricated the grating by mechanical machining of high-density polyethylene. At the camera plane, the +1 and -1 diffraction orders are coherently superimposed, generating an interferogram. We can adjust the shear by selecting the period of the grating and the focal length of the imaging system. We can also alter the direction of the shear by rotating the grating. A gradient-based iterative algorithm is used to reconstruct the wavefront from a set of shear interferograms. The results presented in this study demonstrate the first step towards wavefield sensing in the terahertz band without using a reference wave.

Chocolate inspection by means of phase-contrast imaging using multiple-plane terahertz phase retrieval.

Terahertz (THz) radiation has shown enormous potential for non-destructive inspection in many contexts. Here, we present a method for imaging defects in chocolate bars that can be extended to many other materials. Our method requires only a continuous wave (CW) monochromatic source and detector at relatively low frequencies (280 GHz) corresponding to a relatively long wavelength of 1.1 mm. These components are used to construct a common-path configuration enabling the capturing of several images of THz radiation diffracted by the test object at different axial depths. The captured diffraction-rich images are used to constrain the associated phase retrieval problem enabling full access to the wave field, i.e., real amplitude and phase distributions. This allows full-field diffraction-limited phase-contrast imaging. Thus, we experimentally demonstrate the possibility of identifying contaminant particles with dimensions comparable to the wavelength.

Targeting CD10 on B-Cell Leukemia Using the Universal CAR T-Cell Platform (UniCAR).

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells.

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL-2, TNF and interferon-γ and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells.

Fast 3D form measurement using a tunable lens profiler based on imaging with LED illumination.

We present a fast shape measurement of micro-parts based on depth discrimination in imaging with LED illumination. It is based on a 4f-setup with an electrically adjusted tunable lens at the common Fourier plane. Using such a configuration, the opportunity to implement a fast depth scan by means of a tunable lens without the requirement of mechanically moving parts and depth discrimination using the limited spatial coherence of LED illumination is investigated. The technique allows the use of limited spatially partially coherent illumination which can be easily adapted to the test object by selecting the geometrical parameters of the system accordingly. Using this approach, we demonstrate the approach by measuring the 3D form of a tilted optically rough surface and a cold-formed micro-cup. The approach is robust, fast since required images are captured in less than a second, and eye-safe and offers an extended depth of focus in the range of few millimetres. Using a step height standard, we determine a height error of ±1.75 µm (1σ). This value may be further decreased by lowering the spatial coherence length of the illumination or by increasing the numerical aperture of the imaging system.

Efficient vision ray calibration of multi-camera systems.

Vision ray calibration provides imaging properties of cameras for application in optical metrology by identifying an independent vision ray for each sensor pixel. Due to this generic description of imaging properties, setups of multiple cameras can be considered as one imaging device. This enables holistic calibration of such setups with the same algorithm that is used for the calibration of a single camera. Obtaining reference points for the calculation of independent vision rays requires knowledge of the parameters of the calibration setup. This is achieved by numerical optimization which comes with high computational effort due to the large amount of calibration data. Using the collinearity of reference points corresponding to individual sensor pixels as the measure of accuracy of system parameters, we derived a cost function that does not require explicit calculation of vision rays. We analytically derived formulae for gradient and Hessian matrix of this cost function to improve computational efficiency of vision ray calibration. Fringe projection measurements using a holistically vision ray calibrated system of two cameras demonstrate the effectiveness of our approach. To the best of our knowledge, neither any explicit description of vision ray calibration calculations nor the application of vision ray calibration in holistic camera system calibration can be found in literature.

Γ-profilometry: a new paradigm for precise optical metrology.

We show that the shape of a surface can be unambiguously determined from investigating the coherence function of a wave-field reflected by the surface and without the requirement of a reference wave. Spatio-temporal sampling facilitates the identification of temporal shifts of the coherence function that correspond to finite height differences of the surface. Evaluating these finite differences allows for the reconstruction of the surface using a numerical integration procedure. Spatial sampling of the coherence function is provided by a shear interferometer whereas temporal sampling is achieved by means of a Soleil-Babinet compensator. This low coherence profiling method allows to determine the shape of an object with sub-micrometer resolution and over a large unambiguity range, although it does not require any isolation against mechanical vibration. The approach therefore opens up a new avenue for precise, rugged optical metrology suitable for industrial in-line applications.

And Yet It Moves: Oxidation of the Nuclear Autoantigen La/SS-B Is the Driving Force for Nucleo-Cytoplasmic Shuttling.

Decades ago, we and many other groups showed a nucleo-cytoplasmic translocation of La protein in cultured cells. This shuttling of La protein was seen after UV irradiation, virus infections, hydrogen peroxide exposure and the Fenton reaction based on iron or copper ions. All of these conditions are somehow related to oxidative stress. Unfortunately, these harsh conditions could also cause an artificial release of La protein. Even until today, the shuttling and the cytoplasmic function of La/SS-B is controversially discussed. Moreover, the driving mechanism for the shuttling of La protein remains unclear. Recently, we showed that La protein undergoes redox-dependent conformational changes. Moreover, we developed anti-La monoclonal antibodies (anti-La mAbs), which are specific for either the reduced form of La protein or the oxidized form. Using these tools, here we show that redox-dependent conformational changes are the driving force for the shuttling of La protein. Moreover, we show that translocation of La protein to the cytoplasm can be triggered in a ligand/receptor-dependent manner under physiological conditions. We show that ligands of toll-like receptors lead to a redox-dependent shuttling of La protein. The shuttling of La protein depends on the redox status of the respective cell type. Endothelial cells are usually resistant to the shuttling of La protein, while dendritic cells are highly sensitive. However, the deprivation of intracellular reducing agents in endothelial cells makes endothelial cells sensitive to a redox-dependent shuttling of La protein.

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.

Multiple Aperture Shear-Interferometry (MArS): a solution to the aperture problem for the form measurement of aspheric surfaces.

Multiple Aperture Shear-Interferometry (MArS) is a shape measurement technique that uses multi-spot illumination to overcome the problem of a limited observation aperture of conventional interferometric techniques and thus considerably simplifies the measurement of optical aspheres and freeform surfaces. Using a shear interferometry setup, MArS measures the coherence function in order to obtain wave vector distributions created from multi-spot LED illumination reflected by the specimen. Based on the wave vectors we reconstruct the surface topography of aspheric lenses using an inverse ray tracing approach and prior knowledge about the individual source locations. We present the topographic measurement of two aspheric lenses with different global curvature radii measured with the same identical reflection setup. In addition, we examine the achievable accuracy of the wave vector measurement using a single light source to find physical limits of MArS.

Fast form measurements using a digital micro-mirror device in imaging with partially coherent illumination.

We present a new technique for fast form measurement based on imaging with partially coherent illumination. It consists of a 4f-imaging system with a digital micro-mirror device (DMD) located in the Fourier plane of its two lenses. The setup benefits from spatially partially coherent illumination that allows for depth discrimination and a DMD that enables a fast depth scan. Evaluating the intensity contrast, the 3D form of an object is reconstructed. We show that the technique additionally offers extended depth of focus imaging in microscopy and short measurement times of less than a second.

Versatile Bispidine-Based Bifunctional Chelators for 64 CuII -Labelling of Biomolecules.

Bifunctional chelators as parts of modular metal-based radiopharmaceuticals are responsible for stable complexation of the radiometal ion and for covalent linkage between the complex and the targeting vector. To avoid loss of complex stability, the bioconjugation strategy should not interfere with the radiometal chelation by occupying coordinating groups. The C9 position of the very stable CuII chelator 3,7-diazabicyclo[3.3.1]nonane (bispidine) is virtually predestined to introduce functional groups for facile bioconjugation as this functionalisation does not disturb the metal binding centre. We describe the preparation and characterisation of a set of novel bispidine derivatives equipped with suitable functional groups for diverse bioconjugation reactions, including common amine coupling strategies (bispidine-isothiocyanate) and the Cu-free strain-promoted alkyne-azide cycloaddition. We demonstrate their functionality and versatility in an exemplary way by conjugation to an antibody-based biomolecule and validate the obtained conjugate in vitro and in vivo.

Ultrasmall silicon nanoparticles as a promising platform for multimodal imaging.

Bimodal systems for nuclear and optical imaging are currently being intensively investigated due to their comparable detection sensitivity and the complementary information they provide. In this perspective, we have implemented both modalities on biocompatible ultrasmall silicon nanoparticles (Si NPs). Such nanoparticles are particularly interesting since they are highly biocompatible, have covalent surface functionalization and demonstrate very fast body clearance. We prepared monodisperse citrate-stabilized Si NPs (2.4 ± 0.5 nm) with more than 40 accessible terminal amino groups per particle and, for the first time, simultaneously, a near-infrared dye (IR800-CW) and a radiolabel (64Cu-NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid) have been covalently linked to the surface of such Si NPs. The obtained nanomaterials have been fully characterized using HR-TEM, XPS, UV-Vis and FT-IR spectroscopy. These dual-labelled particles do not exhibit any cytotoxicity in vitro. In vivo studies employing both positron emission tomography (PET) and optical imaging (OI) techniques revealed rapid renal clearance of dual-labelled Si NPs from mice.

Conventional CARs versus modular CARs.

The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.