Generation of integer and fractional vortex beams based on liquid crystal electronically reconfigurable spiral phase plates.

The manufacturing and characterization of a large-size 72-electrode liquid crystal-based reconfigurable spiral phase plate (SPP) is presented. The SPP is addressed by a custom-made driver with 72 independent channels, which allows for the generation of any arbitrary integer or fractional optical vortex beam with topological charges ranging from -24 to +24. The 25 mm diameter device is fabricated using direct laser writing, leading to a fill factor over 99%. The device performance and flexibility exceed previous transparent reconfigurable SPP in terms of size, tuning range, and fill factor. The device and the light path have been simulated using the angular spectrum propagation method, showing excellent correspondence.

ScipionTomo: Towards cryo-electron tomography software integration, reproducibility, and validation.

Image processing in cryogenic electron tomography (cryoET) is currently at a similar state as Single Particle Analysis (SPA) in cryogenic electron microscopy (cryoEM) was a few years ago. Its data processing workflows are far from being well defined and the user experience is still not smooth. Moreover, file formats of different software packages and their associated metadata are not standardized, mainly since different packages are developed by different groups, focusing on different steps of the data processing pipeline. The Scipion framework, originally developed for SPA (de la Rosa-Trevín et al., 2016), has a generic python workflow engine that gives it the versatility to be extended to other fields, as demonstrated for model building (Martínez et al., 2020). In this article, we provide an extension of Scipion based on a set of tomography plugins (referred to as ScipionTomo hereafter), with a similar purpose: to allow users to be focused on the data processing and analysis instead of having to deal with multiple software installation issues and the inconvenience of switching from one to another, converting metadata files, managing possible incompatibilities, scripting (writing a simple program in a language that the computer must convert to machine language each time the program is run), etcetera. Additionally, having all the software available in an integrated platform allows comparing the results of different algorithms trying to solve the same problem. In this way, the commonalities and differences between estimated parameters shed light on which results can be more trusted than others. ScipionTomo is developed by a collaborative multidisciplinary team composed of Scipion team engineers, structural biologists, and in some cases, the developers whose software packages have been integrated. It is open to anyone in the field willing to contribute to this project. The result is a framework extension that combines the acquired knowledge of Scipion developers in close collaboration with third-party developers, and the on-demand design of functionalities requested by beta testers applying this solution to actual biological problems.

Rapid detection of pathogens using lyotropic liquid crystals.

Lyotropic liquid crystals play an important role in many biological environments, such as micelles, liposomes, and phospholipid bilayers of cell membranes. In this work, we explore the performance of lyotropic liquid crystals as biosensors for macromolecules, proteins and whole microorganisms in hydrophilic media, i.e., the natural media where these specimens exist. The aim is to detect specific targets employing simple, unpowered sensors that can be used in the field, with minimum additional equipment. A number of different structures have been explored. The novelty in this work is the inclusion of a new optical effect, flow enhanced amplification, that allows for the semiquantitative detection of microscopic targets in lyotropic liquid crystal cells using the naked eye only.

Blind estimation of DED camera gain in Electron Microscopy.

The introduction of Direct Electron Detector (DED) videos in the Electron Microscope field has boosted Single Particle Analysis to a point in which it is currently considered to be a key technique in Structural Biology. In this article we introduce an approach to estimate the DED camera gain at each pixel from the movies themselves. This gain is needed to have the set of recorded frames into a coherent gray level range, homogeneous over the whole image. The algorithm does not need any other input than the DED movie itself, being capable of providing an estimate of the camera gain image, helping to identify dead pixels and cases of incorrectly calibrated cameras. We propose the algorithm to be used either to validate the experimentally acquired gain image (for instance, to follow its possible change over time) or to verify that there is no residual gain image after experimentally correcting for the camera gain. We show results for a number of DED camera models currently in use (DE, Falcon II, Falcon 3, and K2).

Local analysis of strains and rotations for macromolecular electron microscopy maps.

Macromolecular complexes perform their physiological functions by local rearrangements of their constituents and biochemically interacting with their reaction partners. These rearrangements may involve local rotations and the induction of local strains causing different mechanical efforts and stretches at the different areas of the protein. The analysis of these local deformations may reveal important insight into the way proteins perform their tasks. In this paper we introduce a method to perform this kind of local analysis using Electron Microscopy volumes in a fully objective and automatic manner. For doing so, we exploit the continuous nature of the result of an elastic image registration using B-splines as its basis functions. We show that the results obtained by the new automatic method are consistent with previous observations on these macromolecules.

Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.

In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a workflow-based approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from different programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es.

Nematic liquid crystal reorientation around multi-walled carbon nanotubes mapped via Raman microscopy.

We have studied the formation of topological defects in liquid crystal (LC) matrices induced by multiwalled carbon nanotubes (MWCNTs) and external electric fields. The defects are ascribable to a distortion of the LC molecular director in proximity of the MWCNT surface. The system is analyzed macroscopically using spectroscopic variable angle ellipsometry. Concurrently, confocal micro-Raman spectroscopy is used to study the system state at the microscale. This allows to acquire a three-dimensional, spatially-resolved map of the topological defect, determining scale length variations and orientation topography of the LC molecules around the MWCNT.

A statistical approach to the initial volume problem in Single Particle Analysis by Electron Microscopy.

Cryo Electron Microscopy is a powerful Structural Biology technique, allowing the elucidation of the three-dimensional structure of biological macromolecules. In particular, the structural study of purified macromolecules -often referred as Single Particle Analysis(SPA)- is normally performed through an iterative process that needs a first estimation of the three-dimensional structure that is progressively refined using experimental data. It is well-known the local optimisation nature of this refinement, so that the initial choice of this first structure may substantially change the final result. Computational algorithms aiming to providing this first structure already exist. However, the question is far from settled and more robust algorithms are still needed so that the refinement process can be performed with sufficient guarantees. In this article we present a new algorithm that addresses the initial volume problem in SPA by setting it in a Weighted Least Squares framework and calculating the weights through a statistical approach based on the cumulative density function of different image similarity measures. We show that the new algorithm is significantly more robust than other state-of-the-art algorithms currently in use in the field. The algorithm is available as part of the software suite Xmipp (http://xmipp.cnb.csic.es) and Scipion (http://scipion.cnb.csic.es) under the name "Significant".

Tunable liquid crystal cylindrical micro-optical array for aberration compensation.

A tunable aberration compensation device for rectangular micro-optical systems is proposed and demonstrated. This device, which is based in nematic liquid crystal and a micro-electrode structure, forms gradients in the index of refraction as a function of voltage. We have developed a fringe skeletonizing application in order to extract the 3D wavefront from an interference pattern. This software tool obtains the optical aberrations using Chebyshev polynomials. By using phase shifted electrical signals the aberrations can be controlled independently. A complete independent control over the spherical and coma aberration has been demonstrated. Also, an independent control over the astigmatism aberration has been demonstrated in a broad range. This device has promising applications where aberration compensation is required. The independent compensation achieved for some coefficients, such as astigmatism for example, is more than 2.4 waves.

Three-dimensional reconstruction methods in Single Particle Analysis from transmission electron microscopy data.

The Transmission Electron Microscope provides two-dimensional (2D) images of the specimens under study. However, the architecture of these specimens is defined in a three-dimensional (3D) coordinate space, in volumetric terms, making the direct microscope output somehow "short" in terms of dimensionality. This situation has prompted the development of methods to quantitatively estimate 3D volumes from sets of 2D images, which are usually referred to as "three-dimensional reconstruction methods". These 3D reconstruction methods build on four considerations: (1) The relationship between the 2D images and the 3D volume must be of a particularly simple type, (2) many 2D images are needed to gain 3D volumetric information, (3) the 2D images and the 3D volume have to be in the same coordinate reference frame and (4), in practical terms, the reconstructed 3D volume will only be an approximation to the original 3D volume which gave raise to the 2D projections. In this work we will adopt a quite general view, trying to address a large community of interested readers, although some sections will be particularly devoted to the 3D analysis of isolated macromolecular complexes in the application area normally referred to as Single Particle Analysis (SPA).

A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs.

MOTIVATION: Structural information of macromolecular complexes provides key insights into the way they carry out their biological functions. Achieving high-resolution structural details with electron microscopy requires the identification of a large number (up to hundreds of thousands) of single particles from electron micrographs, which is a laborious task if it has to be manually done and constitutes a hurdle towards high-throughput. Automatic particle selection in micrographs is far from being settled and new and more robust algorithms are required to reduce the number of false positives and false negatives. RESULTS: In this article, we introduce an automatic particle picker that learns from the user the kind of particles he is interested in. Particle candidates are quickly and robustly classified as particles or non-particles. A number of new discriminative shape-related features as well as some statistical description of the image grey intensities are used to train two support vector machine classifiers. Experimental results demonstrate that the proposed method: (i) has a considerably low computational complexity and (ii) provides results better or comparable with previously reported methods at a fraction of their computing time. AVAILABILITY: The algorithm is fully implemented in the open-source Xmipp package and downloadable from http://xmipp.cnb.csic.es.

FASTDEF: fast defocus and astigmatism estimation for high-throughput transmission electron microscopy.

In this work we present a fast and automated algorithm for estimating the contrast transfer function (CTF) of a transmission electron microscope. The approach is very suitable for High Throughput work because: (a) it does not require any initial defocus estimation, (b) it is almost an order of magnitude faster than existing approaches, (c) it opens the way to well-defined extensions to the estimation of higher order aberrations, at the same time that provides defocus and astigmatism estimations comparable in accuracy to well established methods, such as Xmipp and CTFFIND3 approaches. The new algorithm is based on obtaining the wrapped modulating phase of the power spectra density pattern by the use of a quadrature filter. This phase is further unwrapped in order to obtain the continuous and smooth absolute phase map; then a Zernike polynomial fitting is performed and the defocus and astigmatism parameters are determined. While the method does not require an initial estimation of the defocus parameters or any non-linear optimization procedure, these approaches can be used if further refinement is desired. Results of the CTF estimation method are presented for standard negative stained images, cryo-electron microscopy images in the absence of carbon support, as well as micrographs with only ice. Additionally, we have also tested the proposed method with micrographs acquired from tilted and untilted samples, obtaining good results. The algorithm is freely available as a part of the Xmipp package [http://xmipp.cnb.csic.es].

Xmipp 3.0: an improved software suite for image processing in electron microscopy.

Xmipp is a specialized software package for image processing in electron microscopy, and that is mainly focused on 3D reconstruction of macromolecules through single-particles analysis. In this article we present Xmipp 3.0, a major release which introduces several improvements and new developments over the previous version. A central improvement is the concept of a project that stores the entire processing workflow from data import to final results. It is now possible to monitor, reproduce and restart all computing tasks as well as graphically explore the complete set of interrelated tasks associated to a given project. Other graphical tools have also been improved such as data visualization, particle picking and parameter "wizards" that allow the visual selection of some key parameters. Many standard image formats are transparently supported for input/output from all programs. Additionally, results have been standardized, facilitating the interoperation between different Xmipp programs. Finally, as a result of a large code refactoring, the underlying C++ libraries are better suited for future developments and all code has been optimized. Xmipp is an open-source package that is freely available for download from: http://xmipp.cnb.csic.es.

Validation of electrodeposited 241Am alpha-particle sources for use in liquified gas detectors at cryogenic temperatures.

This paper describes a procedure for the validation of alpha-particle sources (exempt unsealed sources) to be used in experimental setups with liquefied gases at cryogenic temperatures (down to -196 °C) and high vacuum. These setups are of interest for the development and characterization of neutrino and dark matter detectors based on liquid argon, among others. Due to the high purity requirements, the sources have to withstand high vacuum and cryogenic temperatures for extended periods. The validation procedure has been applied to 241Am sources produced by electrodeposition.

Identification of dyes and matrices for dye doped polymer waveguide emitters covering the visible spectrum.

Polymer based photonic devices offer the possibility cost effective roll-to-roll manufacture of photonic devices. The incorporation of luminescent dopants within a solid polymer waveguide allows for the generation of light within the device avoiding tedious mechanical light coupling. However, when a dopant is embedded in a solid matrix, depending on its concentration and the nature of materials involved, the emitted light may be quenched due to aggregation effects. In this work, thin films and ridge waveguides processed by UV-photolithography have been successfully obtained from a selection of standard photopolymerizable organic monomers, SU8, EpoCore and OrmoStamp doped with a selection of standard dyes like Rhodamine-B, Coumarin-540A and Pyrromethene-580. All structures were manufactured on glass substrates. An analysis of the solubility and optical properties including band gap energy, absorption coefficient ([Formula: see text]) and fluorescence of the doped photoresists at different concentrations has been performed. Photoresists doped with Rhodamine-B shows a higher energy of indirect allowed band gap transition (2.04-2.09 eV) compared to previously reported pure Rhodamine-B thin films (1.95-1.98 eV). Fabrication protocols of dye doped photoresists covering the entire visible spectrum is established.

Measurement of local resolution in electron tomography.

Resolution (global and local) is one of the most reported metrics of quality measurement in Single Particle Analysis (SPA). However, in electron tomography, the situation is different and its computation is not straightforward. Typically, resolution estimation is global and, therefore, reduces the assessment of a whole tomogram to a single number. However, it is known that tomogram quality is spatially variant. Still, up to our knowledge, a method to estimate local quality metrics in tomography is lacking. This work introduces MonoTomo, a method developed to estimate locally in a tomogram the highest reliable frequency component, expressed as a form of local resolution. The fundamentals lie in a local analysis of the density map via monogenic signals, which, in analogy to MonoRes, allows for local estimations. Results with experimental data show that the local resolution range that MonoTomo casts agrees with reported resolution values for experimental data sets, with the advantage of providing a local estimation. A range of applications of MonoTomo are suggested for further exploration.

The chaperonin CCT controls T cell receptor-driven 3D configuration of centrioles.

T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, Δ1 and Δ2) of αß-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.

Dynamic multilevel spiral phase plate generator.

The design and characterisation of a reconfigurable multi-level spiral phase plate is shown. The device is based on a pie-shape liquid-crystal structure with 24 slices driven by custom electronics that allow independent excitation control of each electrode. The electrooptical cell was manufactured using maskless laser ablation lithography and has shown an unprecedented high fill factor. The topological charge can be dynamically changed between 1, 2, 3, 4, 6, 8 and 12. The device has been calibrated and characterised at 632.8 nm but can be employed at any wavelength in the visible and near infrared spectrum, just modifying the driving parameters of the electrodes. The experimental results have been compared to predictions derived from simulations. An excellent correspondence between theoretical and experimental result has been found in all cases.

A review of resolution measures and related aspects in 3D Electron Microscopy.

Fourier Shell Correlation, Spectral Signal-to-Noise Ratio, Fourier Neighbour Correlation, and Differential Phase Residual are different measures that have been proposed over time to determine the spatial resolution achieved by a certain 3D reconstruction. Estimates of B-factors to describe the reduction in signal-to-noise ratio with increasing resolution is also a useful parameter. All these concepts are interrelated and different thresholds have been given for each one of them. However, the problem of resolution assessment in 3DEM is still far from settled and preferences are normally adopted in order to choose the "correct" threshold. In this paper we review the different concepts, their theoretical foundations and the derivation of their statistical distributions (the basis for establishing sensible thresholds). We provide theoretical justifications for some common practices in the field for which a formal justification was missing. We also analyze the relationship between SSNR and B-factors, the electron dose needed for achieving a given contrast and resolution, the number of images required, etc. Finally, we review the consequences for the number of particles needed to achieve a certain resolution and how to analyze the Signal-to-Noise Ratio for a sequence of imaging operations.

A Survey of the Use of Iterative Reconstruction Algorithms in Electron Microscopy.

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET).