3D nanoprinted catadioptric fiber sensor for dual-axis distance measurement during vitrectomy.

Retinal damage is a common intraoperative complication during vitrectomy, caused by a complex interplay between the suction of the vitrectome, the cut- and aspiration rate, and the distance of the instrument to the retina. To control this last factor, we developed two miniaturized fiber-optic distance sensors based on low-coherence interferometry for direct integration into the vitrectome. Both sensors have a diameter of 250 µm, which makes them compatible with a 25G vitrectome. The first sensor measures distance in the lateral direction. The second sensor is capable of simultaneously measuring distance in both the lateral and the axial direction. Axial and lateral directions correspond to the direction of the cutter port of the vitrectome and the direction along the vitrectome's shaft, respectively. In both sensors, a free-form mirror deflects and focuses the beam in the lateral direction. In the dual-axis distance sensor, an additional lens is integrated into the free-form mirror for distance measurement in the axial direction. The beam-shaping micro-optics at the tip of the sensor fibers were fabricated through two-photon polymerization and are selectively gold coated for increased reflectivity of the mirror. Distance measurements were successfully demonstrated in artificial samples and in ex vivo pig eyes with a back-end that uses a current-tuned VCSEL as a swept-source. We experimentally demonstrate that the complete sensor system can attain a S N R max of up to 80 dB. The small dimensions of the developed sensors make them a potential solution for various other medical applications.

Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling.

G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19 F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution.

Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible ßγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including ßγ-dimers previously thought to be nonexistent, except for combinations containing ß5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1ßγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli.

Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization.

Fresnel transform as a projection onto a Nijboer-Zernike basis set.

The Fresnel transform is widely used in optics to calculate the free-space propagation of paraxial fields. Generally, there is no analytical solution for the Fresnel transform; therefore, the numerical methods are used often. In this Letter, we propose a new semi-analytical method to calculate the Fresnel transform, which is based on an extended Nijboer-Zernike theory. We calculate two examples to investigate how the sampling rate and maximal number of Zernike polynomials affect the accuracy of our results, and then use this method to calculate the reconstruction of two different kinds of holograms. At the end, we discuss the advantages and disadvantages of our method.

Transgenic mice for cGMP imaging.

RATIONALE: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. OBJECTIVE: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. METHODS AND RESULTS: Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken ß-actin/ß-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. CONCLUSIONS: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

Chromatic confocal matrix sensor with actuated pinhole arrays.

We present two versions of a chromatic confocal matrix sensor for the snapshot acquisition of three-dimensional objects. The first version contains separate illumination and detection pinhole arrays, while the second version uses a single pinhole array in double pass. The discrete lateral measurement points defined by the illumination and detection pinhole arrays are evaluated in parallel with a hyperspectral detection module. As this approach enables the spectrometric evaluation of all lateral channels, multilayer objects can be analyzed. To increase the lateral resolution the pinhole arrays are moved by micromechanical actuators. The paper includes a quantitative evaluation of the chromatic confocal module and proof-of-principle experiments with the full sensor system.

Numerical solution of nonparaxial scalar diffraction integrals for focused fields.

In this paper, we present sampling conditions for fast-Fourier-transform-based field propagations. The input field and the propagation kernel are analyzed in a combined manner to derive sampling criteria that guarantee accurate calculation results in the output plane. These sampling criteria are also applicable to the propagation of general fields. For focal field calculations, geometrical optics is used to obtain a priori knowledge about the input and output fields. This a priori knowledge is used to determine an optimum balance between computational load and calculation accuracy. In a numerical example, correct results are obtained even though both the input field and the propagation kernel are sampled below the Nyquist rate. Finally, we show how chirp z-transform-based zoom-algorithms may be analyzed using the same techniques.

Fast nonparaxial scalar focal field calculations.

An efficient algorithm for calculating nonparaxial scalar field distributions in the focal region of a lens is discussed. The algorithm is based on fast Fourier transform implementations of the first Rayleigh-Sommerfeld diffraction integral and assumes that the input field at the pupil plane has a larger extent than the field in the focal region. A sampling grid is defined over a finite region in the output plane and referred to as a tile. The input field is divided into multiple separate spatial regions of the size of the output tile. Finally, the input tiles are added coherently to form a summed tile, which is propagated to the output plane. Since only a single tile is propagated, there are significant reductions of computational load and memory requirements. This method is combined either with a subpixel sampling technique or with a chirp z-transform to realize smaller sampling intervals in the output plane than in the input plane. For a given example the resulting methods enable a speedup of approximately 800× in comparison to the normal angular spectrum method, while the memory requirements are reduced by more than 99%.

Aberration analysis of optimized Alvarez-Lohmann lenses.

In this paper aberrations in Alvarez-Lohmann lenses are analyzed, and a semi-analytical strategy for compensation is derived. An x-y polynomial model is used to describe the aberrations and classify them into static and dynamic components. The lenses are enhanced by higher-order polynomials, and a numerical optimization process is used to determine the most influential coefficients. Two simulations of corrected systems are presented. The first one is optimized for on-axis imaging. The second system is optimized for multiple field points and shows the limitations of a single Alvarez-Lohmann lens. Two systems overcoming these limitations by introducing additional optical surfaces are presented, and their performance is analyzed in simulations.

Spectral characteristics of chromatic confocal imaging systems.

We present signal-generation models for chromatic confocal imaging systems with illumination and detection pinholes of finite size: a collinear model that considers neither aberrations nor diffraction effects, a geometrical model that accounts for aberrations, and a wave optical model covering both aberrations and diffraction effects. These models are aimed at describing the spectral response of multipoint sensor systems with field-dependent aberrations and vignetting effects. They are suitable for single- and double-pass systems with either diffusely or specularly reflecting surfaces under test. We show experimental results to verify our models.

Stereo reconstruction from microscopic images for computer-assisted ophthalmic surgery.

PURPOSE: This work presents a novel platform for stereo reconstruction in anterior segment ophthalmic surgery to enable enhanced scene understanding, especially depth perception, for advanced computer-assisted eye surgery by effectively addressing the lack of texture and corneal distortions artifacts in the surgical scene. METHODS: The proposed platform for stereo reconstruction uses a two-step approach: generating a sparse 3D point cloud from microscopic images, deriving a dense 3D representation by fitting surfaces onto the point cloud, and considering geometrical priors of the eye anatomy. We incorporate a pre-processing step to rectify distortion artifacts induced by the cornea's high refractive power, achieved by aligning a 3D phenotypical cornea geometry model to the images and computing a distortion map using ray tracing. RESULTS: The accuracy of 3D reconstruction is evaluated on stereo microscopic images of ex vivo porcine eyes, rigid phantom eyes, and synthetic photo-realistic images. The results demonstrate the potential of the proposed platform to enhance scene understanding via an accurate 3D representation of the eye and enable the estimation of instrument to layer distances in porcine eyes with a mean average error of 190  µ m , comparable to the scale of surgeons' hand tremor. CONCLUSION: This work marks a significant advancement in stereo reconstruction for ophthalmic surgery by addressing corneal distortions, a previously often overlooked aspect in such surgical scenarios. This could improve surgical outcomes by allowing for intra-operative computer assistance, e.g., in the form of virtual distance sensors.

Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Tunable hyperchromatic lens system for confocal hyperspectral sensing.

A new approach for confocal hyperspectral sensing based on the combination of a diffractive optical element and a tunable membrane fluidic lens is demonstrated. This highly compact lens system is designed to maximize the longitudinal chromatic aberration and select a narrow spectral band by spatial filtering. Changing the curvature of the fluidic lens allows the selected band to be scanned over the whole given spectrum. A hybrid prototype with an integrated electro-magnetic micro-actuator has been realized to demonstrate the functionality of the system. Experimental results show that the spectrum transmitted by the system can be tuned over the entire visible wavelength range, from 450 to 900 nm with a narrow and almost constant linewidth of less than 15 nm. Typical response time for scanning the spectrum by 310 nm is less than 40 ms and the lens system shows a highly linear relationship with the driving current.

Spectrally multiplexed chromatic confocal multipoint sensing.

We present a concept for chromatic confocal distance sensing that employs two levels of spectral multiplexing for the parallelized evaluation of multiple lateral measurement points; at the first level, the chromatic confocal principle is used to encode distance information within the spectral distribution of the sensor signal. For lateral multiplexing, the total spectral bandwidth of the sensor is split into bands. Each band is assigned to a different lateral measurement point by a segmented diffractive element. Based on this concept, we experimentally demonstrate a chromatic confocal three-point sensor that is suitable for harsh production environments, since it works with a single-point spectrometer and does not require scanning functionality. The experimental system has a working distance of more than 50 mm, a measurement range of 9 mm, and an axial resolution of 50 µm.

Optical performance of coherent and incoherent imaging systems in the presence of ghost images.

Ghost image analysis is of interest for optical designers as ghost images may degrade image quality in imaging systems. In a previous paper by Abd El-Maksoud and Sasian [Appl. Opt. 50, 2305 (2011)10.1364/AO.50.002305], ghost image analysis for incoherent systems was evaluated using geometrical optics. Some criteria were presented to identify focused ghost images at the nominal image plane. The main goal of this paper is to provide a conceptual understanding of ghost image formation and its impact on the performance of imaging systems using wave theory and Fourier optics. To achieve this goal, a methodology is developed to model ghost images after considering diffraction effects. Expressions for the ghost diffraction point spread function and the ghost transfer function are presented. These functions are used to construct effective point spread and effective transfer functions. To provide insights on the developed theory, some simulation examples are provided and discussed.

Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.

Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.

Vascular endothelial growth factor gene therapy improves nerve regeneration in a model of obstetric brachial plexus palsy.

The treatment of obstetric brachial plexus palsy has been limited to conservative therapies and surgical reconstruction of peripheral nerves. In addition to the damage of the brachial plexus itself, it also leads to a loss of the corresponding motoneurons in the spinal cord, which raises the need for supportive strategies that take the participation of the central nervous system into account. Based on the protective and regenerative effects of VEGF on neural tissue, our aim was to analyse the effect on nerve regeneration by adenoviral gene transfer of vascular endothelial growth factor (VEGF) in postpartum nerve injury of the brachial plexus in rats. In the present study, we induced a selective crush injury to the left spinal roots C5 and C6 in 18 rats within 24 hours after birth and examined the effect of VEGF-gene therapy on nerve regeneration. For gene transduction an adenoviral vector encoding for VEGF165 (AdCMV.VEGF165) was used. In a period of 11 weeks, starting 3 weeks post-operatively, functional regeneration was assessed weekly by behavioural analysis and force measurement of the upper limb. Morphometric evaluation was carried out 8 months post-operatively and consisted of a histological examination of the deltoid muscle and the brachial plexus according to defined criteria of degeneration. In addition, atrophy of the deltoid muscle was evaluated by weight determination comparing the left with the right side. VEGF expression in the brachial plexus was quantified by an enzyme-linked immunosorbent assay (ELISA). Furthermore the motoneurons of the spinal cord segment C5 were counted comparing the left with the right side. On the functional level, VEGF-treated animals showed faster nerve regeneration. It was found less degeneration and smaller mass reduction of the deltoid muscle in VEGF-treated animals. We observed significantly less degeneration of the brachial plexus and a greater number of surviving motoneurons (P < 0·05) in the VEGF group. The results of this study confirmed the positive effect of VEGF-gene therapy on regeneration and survival of nerve cells. We could demonstrate a significant improvement on the motor-functional as well as on the histomorphological level. However, increased vascularization of the nerve tissue caused by VEGF does not seem to be the major reason for these effects. The clinical use of adenoviral VEGF-gene therapy in the newborn cannot be justified so far.

Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.