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The longstanding study of gross anatomy experienced a considerable improvement with the advent of the microscope in the early 17th century. The representative personality of this new era certainly was Marcello Malpighi, seen as “founder of microscopic anatomy”. He studied, with a rudimentary compound microscope, numerous tissues and organs of several classes of animals, as well as plants. He described, for the first time, the microscopic structure of the nervous system, identifying in the gray matter of its various levels minute elements he took as “glands”. It should be reminded that the concept of “cell” (and “nerve cell”) was unknown at his time. Many researchers followed, performing microscopic studies, but without better results, and Malpighi’s view was maintained until the beginning of the 19th century, when new histological processing and staining techniques appeared, as well as improved microscopes. RESUMO O estudo de longa data da anatomia macroscópica experimentou um incremento considerável com o advento do microscópio no início do século 17. A personalidade representativa dessa nova era foi, certamente, Marcello Malpighi, considerado “fundador da anatomia microscópica”. Ele estudou, com um microscópio composto rudimentar, numerosos tecidos e órgãos de diversas classes de animais, assim como plantas. Descreveu, pela primeira vez, a estrutura microscópica do sistema nervoso, identificando na substância cinzenta dos vários níveis elementos de minúsculas dimensões, que denominou “glândulas”. Deve-se lembrar que o conceito de “célula” (e de “célula nervosa”) era desconhecido naquele tempo. Muitos pesquisadores seguiram realizando estudos microscópicos, mas sem resultados melhores, e o entendimento de Malpighi foi mantido até o início do século 19, quando apareceram técnicas histológicas novas de processamento e de coloração, assim como microscópios mais aprimorados.

Lensless microscopy requires the simplest possible configuration, as it uses only a light source, the sample and an image sensor. The smallest practical microscope is demonstrated here. In contrast to standard lensless microscopy, the object is located near the lighting source. Raster optical microscopy is applied by using a single-pixel detector and a microdisplay. Maximum resolution relies on reduced LED size and the position of the sample respect the microdisplay. Contrarily to other sort of digital lensless holographic microscopes, light backpropagation is not required to reconstruct the images of the sample. In a mm-high microscope, resolutions down to 800 nm have been demonstrated even when measuring with detectors as large as 138 μm × 138 μm, with field of view given by the display size. Dedicated technology would shorten measuring time., This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 737089 and from the Ministry of Science and Innovation, the Spanish State Research Agency and the European Regional Development Fund through project PID2019-105714RB-I00.

La trazabilidad en mediciones superficiales de micro y nanoestructuras es condición in-eludible para garantizar la calidad en proyectos de innovación, investigación y desarrollo en el espectro disciplinar que abarca las ciencias químicas, biológicas, de materiales, siendo además un requisito para mediciones de rugosidad en la industria. La confiabilidad metro-lógica (ISO 10012) presupone el uso de instrumentos calibrados, patrones trazables, procedimientos de medición validados y balances de incertidumbre. Estos requerimientos no son habituales en microscopía y representa una debilidad a subsanar. En este trabajo se presentan los resultados de la intercomparación de mediciones de textura superficial en patrones 2D y 3D con cuatro tipos de instrumentos: Rugosímetro, Microscopio Confocal, Microscopio de Fuerza Atómica y Microscopio Electrónico de Barrido. Las evaluaciones de conformidad (ISO 17043) mediante la comparación de los errores normalizados (En), que ponderan los efectos sistemáticos y sus incertidumbres, convalidan el uso de los microscopios como instrumentos metrológicos. Fil: Brambilla, Nancy. Universidad Tecnológica Nacional. Facultad Regional Córdoba; Argentina. Universidad Tecnológica Nacional. Facultad Regional Córdoba. Centro de Investigación y Transferencia En Metrología; Argentina Fil: Brusa, Daniel Horacio. Universidad Tecnológica Nacional. Facultad Regional Córdoba; Argentina. Universidad Tecnológica Nacional. Facultad Regional Córdoba. Centro de Investigación y Transferencia En Metrología; Argentina Fil: Caselles, Juan Esteban. Universidad Tecnológica Nacional. Facultad Regional Córdoba; Argentina. Universidad Tecnológica Nacional. Facultad Regional Córdoba. Centro de Investigación y Transferencia En Metrología; Argentina Fil: Mas, Carlos Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Schurrer, Clemar Aldino. Universidad Tecnológica Nacional. Facultad Regional Córdoba; Argentina. Universidad Tecnológica Nacional. Facultad Regional Córdoba. Centro de Investigación y Transferencia En Metrología; Argentina

Dc-dc converters can be found in different kinds of electric vehicles (EVs). Their main function is to accommodate voltages and currents to the motor or other EV systems requirements. The use of wide-bandgap (WBG) devices can improve the efficiency of silicon-based power converters, qualifying also for higher switching frequencies. In this article the features of a dual active bridge (DAB) converter are studied. The high voltage side of the DAB is implemented with Silicon Carbide (SiC) MOSFETs. For the low voltage side two types of devices are used: either Gallium Nitride (GaN) enhancement high-electronmobility transistors (e-HEMTs) or SiC MOSFETs. The influence of switching frequency and output power on the efficiency are evaluated. The parallel connection of GaN devices is proposed to overcome the device current limits and thus increase the overall DAB converter output power. A feedback controller has been designed to reduce the effects on the output voltage of load changes. The DAB converter evaluation has been realized by using MATLAB/Simulink and PLECS software. This work was supported by the Industrial Doctorate Plan of the Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya, and the Ministerio de Ciencia, Innovación y Universidades of Spain within the project PID2019-111420RB-I00.

Imaging confocal microscopy (ICM) and focus variation (FV) are two of the most used technologies for 3D surface metrology. Both methods rely on the depth of focus of the microscope objective, which depends on its numerical aperture and wavelength of the light source to compute an optical section. In this paper we study how several methods of structured illumination microscopy affect the metrological characteristics of an areal optical profiler. We study the effect of the projection of different structured patterns, the sectioning algorithms, and the use of high and low frequency components onto the optically sectioned image. We characterized their performance in terms of system noise, instrument transfer function and metrological characteristics such as roughness parameters and step height values.

Acoustic waves in an optical medium cause rapid periodic changes in the refraction index, leading to diffraction effects. Such acoustically controlled diffraction can be used to modulate, deflect, and focus light at microsecond timescales, paving the way for advanced optical microscopy designs that feature unprecedented spatiotemporal resolution. In this article, we review the operational principles, optical properties, and recent applications of acousto-optic (AO) systems for advanced microscopy, including random-access scanning, ultrafast confocal and multiphoton imaging, and fast inertia-free light-sheet microscopy. As AO technology is reaching maturity, designing new microscope architectures that utilize AO elements is more attractive than ever, providing new exciting opportunities in fields as impactful as optical metrology, neuroscience, embryogenesis, and high-content screening.

The recent advances in chip-size microscopy based on optical scanning with spatially resolved nano-illumination light sources are presented. This new straightforward technique takes advantage of the currently achieved miniaturization of LEDs in fully addressable arrays. These nano-LEDs are used to scan the sample with a resolution comparable to the LED sizes, giving rise to chip-sized scanning optical microscopes without mechanical parts or optical accessories. The operation principle and the potential of this new kind of microscope are analyzed through three different implementations of decreasing LED dimensions from 20 µm down to 200 nm.

Two reactive phenanthrene derivatives, 4-(1H phenanthrol [9,10-d] imidazole-2-yl) benzaldehyde (PIB) and 6,9-dimethoxyphenanthro[9,10-c]furan-1,3-dione (PA) with high fluorescent quantum yields were prepared and used as fluorescent marker in fluorescence microscopy. In particular, silane modified μmZeolite-L containing amino group (-NH2) in the surface were labeled with the phenanthrene derivatives allowing good imaging resolution and spectroscopy measurements. The presence of a large Stokes shift of the probes due to their intramolecular charge-transfer character gives an advantage of the compounds in confocal laser fluorescence microscopy due to easy signal separation in excitation and emission wavelengths. On the other hand, these results open up the possibility of using these probes for visualization of Zeolite-based materials commonly used as catalysts in thermal and photochemical reactions.

In lensless microscopy, spatial resolution is usually provided by the pixel density of current digital cameras, which are reaching a hard-to-surpass pixel size / resolution limit over 1 µm. As an alternative, the dependence of the resolving power can be moved from the detector to the light sources, offering a new kind of lensless microscopy setups. The use of continuously scaled-down Light-Emitting Diode (LED) arrays to scan the sample allows resolutions on order of the LED size, giving rise to compact and low-cost microscopes without mechanical scanners or optical accessories. In this paper, we present the operation principle of this new approach to lensless microscopy, with simulations that demonstrate the possibility to use it for super-resolution, as well as a first prototype. This proof-of-concept setup integrates an 8 × 8 array of LEDs, each 5 × 5 μm2pixel size and 10 μmpitch, and an optical detector. We characterize the system using Electron-Beam Lithography (EBL) pattern. Our prototype validates the imaging principle and opens the way to improve resolution by further miniaturizing the light sources. © 2020 Optical Society of America under the terms of theOSA Open Access Publishing Agreement

Recently, Fourier light field microscopy was proposed to overcome the limitations in conventional light field microscopy by placing a micro-lens array at the aperture stop of the microscope objective instead of the image plane. In this way, a collection of orthographic views from different perspectives are directly captured. When inspecting fluorescent samples, the sensitivity and noise of the sensors are a major concern and large sensor pixels are required to cope with low-light conditions, which implies under-sampling issues. In this context, we analyze the sampling patterns in Fourier light field microscopy to understand to what extent computational super-resolution can be triggered during deconvolution in order to improve the resolution of the 3D reconstruction of the imaged data.

The time scale for interfacial photoinduced electron transfer (PeT) in plasmonic nanoparticles is not well established and the details are still under debate. This has renewed the interest in studying the electron transfer effect from both experimental and theoretical points of view. We present a quantitative analysis of PeT in single spherical gold (Au) and gold@palladium core@shell (Au@Pd) nanoparticles supported on reduced graphene oxide (RGO) using dark-field hyperspectral microscopy (DFHM) and electrochemical impedance spectroscopy (EIS). By studying the plasmon bandwidth in the scattering spectra of single particles and by correlating it to the plasmon damping processes we showed that PeT occurs from the AuNPs to RGO in a 10 fs time scale with a quantum efficiency of 35%. The introduction of a Pd shell on the AuNPs decreases the PeT time, with transfer occurring in as little as 1.7 fs with quantum yield higher than 74%. Furthermore, EIS showed a smaller resistance for PeT on RGO/Au@PdNPs under green light illumination. Our results can improve the understanding of the chemical interface damping process due to PeT in plasmonic nanomaterials and can enable the design of more efficient plasmon enhanced photocatalysts.

Lightfield microscopy has raised growing interest in the last few years. Its ability to get three-dimensional information about the sample in a single shot makes it suitable for many applications in which time resolution is fundamental. In this paper we present a novel device, which is capable of converting any conventional microscope into a lightfield microscope. Based on the Fourier integral microscope concept, we designed the lightfield microscope eyepiece. This is coupled to the eyepiece port, to let the user exploit all the host microscope’s components (objective turret, illumination systems, translation stage, etc.) and get a 3D reconstruction of the sample. After the optical design, a proof-of-concept device was built with off-the-shelf optomechanical components. Here, its optical performances are demonstrated, which show good matching with the theoretical ones. Then, the pictures of different samples taken with the lightfield eyepiece are shown, along with the corresponding reconstructions. We demonstrated the functioning of the lightfield eyepiece and lay the foundation for the development of a commercial device that works with any microscope.

Recently, integral (also known as lightfield or plenoptic) imaging concept has been applied successfully to microscopy. The main advantage of lightfield microscopy when compared with conventional 3D imaging techniques is that it offers the possibility of capturing the 3D information of the sample after a single shot. However, integral microscopy is now facing many challenges, like improving the resolution and depth of field of the reconstructed specimens or the development and optimization of specially-adapted reconstruction algorithms. This contribution is devoted to review a new paradigm in lightfield microscopy, namely, the Fourier-domain integral microscope (FiMic), that improves the capabilities of the technique, and to present recent advances and applications of this new architecture.

Most 3D metrological microscopes used today require a scanning through the optical axis, which is time consuming. The common techniques are Coherence Scanning Interferometry (CSI), Imaging Confocal Microscopy (ICM), and Focus Variation (FV). If one technique is good for smooth surfaces, it is not for rough ones, while the good for rough is too noisy for smooth ones. Additionally, high local slopes are also dependent on the scattering properties of the surface, making the Numerical Aperture of the objective the most important property of the microscope. Imaging Confocal Microscopy is the best compromise in terms of surface application range (from smooth to rough), high local slopes on shiny surfaces, highest numerical aperture and highest possible magnification. Unfortunately, any kind of Confocal microscope today (laser scan, disc scan or microdisplay scan) requires an in-plane scanning to build up the confocal image in addition to the vertical scan, increasing the total measuring time in comparison to CSI and FV. This is against the needs of quality control in production environments, where scanning speed must be as short as possible. In this paper, we use a Microdisplay Scanning Microscope for obtaining the confocal image only relying on a single image per plane. We use a structured illumination to project a desired pattern onto the surface with a very well-defined frequency and direction. By means of the Hilbert transform, we digitally shift the projected pattern one or many times to recover the bright field and the optical sectioned images. This new method reduces significantly the measurement time, simplifies the overall cost of the system and eliminates the maintenance of scanning devices, while maintaining the optical sectioning properties of each plane. We also studied the performance of the resulting topography in terms of system noise, accuracy, repeatability and fidelity of the surface using different methods to obtain the confocal image. Finally, we also compared the results with true confocal results and with other techniques that require a single image per plane, such as Active illumination Focus Variation (AiFV).

We report a protocol that takes advantage of the Fourier lightfield microscopy concept for providing 3D darkfield images of volumetric samples in a single-shot. This microscope takes advantage of the Fourier lightfield configuration, in which a lens array is placed at the Fourier plane of the microscope objective, providing a direct multiplexing of the spatio-angular information of the sample. Using the proper illumination beam, the system collects the light scattered by the sample while the background light is blocked out. This produces a set of orthographic perspective images with shifted spatial-frequency components that can be recombined to produce a 3D darkfield image. Applying the adequate reconstruction algorithm high-contrast darkfield optical sections are calculated in real time. The presented method is applied for fast volumetric reconstructions of unstained 3D samples.

Recent research into miniaturized illumination sources has prompted the development of alternative microscopy techniques. Although they are still being explored, emerging nano-light-emitting-diode (nano-LED) technologies show promise in approaching the optical resolution limit in a more feasible manner. This work presents the exploration of their capabilities with two different prototypes. In the first version, a resolution of less than 1 µm was shown thanks to a prototype based on an optically downscaled LED using an LED scanning transmission optical microscopy (STOM) technique. This research demonstrates how this technique can be used to improve STOM images by oversampling the acquisition. The second STOM-based microscope was fabricated with a 200 nm GaN LED. This demonstrates the possibilities for the miniaturization of on-chip-based microscopes., This work was partially supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 737089—ChipScope.

Surface structural, electronic and electrical properties of the quaternary alloy AlInGaN/GaN heterostructures are investigated. Surface termination, atomic arrangement, electronic and electrical properties of the (0001) surface and (10–11) V-defect facets have been experimentally analyzed using various surface sensitive techniques including spectroscopy and microscopy. Moreover, the effect of sub-band gap (of the barrier layer) illumination on contact potential difference (VCPD) and the role of oxygen chemisorption have been studied.

During the last thirty years, microstructural and technological studies on ceramic glazes have been essentially carried out through the use of Scanning Electron Microscopy (SEM) combined with energy dispersive X-ray analysis (EDX). On the contrary, optical microscopy (OM) has been considered of limited use in solving the very complex and fine-scale microstructures associated with ceramic glazes. As the crystallites formed inside glazes are sub- and micrometric, a common misconception is that it is not possible to study them by OM. This is probably one of the reasons why there are no available articles and textbooks and even no visual resources for describing and characterizing the micro-crystallites formed in glaze matrices. A thin section petrography (TSP) for ceramic glaze microstructures does not exist yet, neither as a field of study nor conceptually. In the present contribution, we intend to show new developments in the field of ceramic glaze petrography, highlighting the potential of OM in the microstructural studies of ceramic glazes using petrographic thin sections. The outcomes not only stress the pivotal role of thin section petrography for the study of glaze microstructures but also show that this step should not be bypassed to achieve reliable readings of the glaze microstructures and sound interpretations of the technological procedures. We suggest the adoption by the scientific community of an alternative vision on glaze microstructures to turn thin section petrography for glaze microstructures into a new specialized petrographic discipline. Such an approach, if intensively developed, has the potential to reduce the time and costs of scientific investigations in this specific domain. In fact, it can provide key reference data for the identification of the crystallites in ceramic glazes, avoiding the repetition of exhaustive protocols of expensive integrated analyses.

Integral microscopy is a 3D imaging technique that permits the recording of spatial and angular information of microscopic samples. From this information it is possible to calculate a collection of orthographic views with full parallax and to refocus computationally, at will, through the 3D specimen. An important drawback of integral microscopy, especially when dealing with thick samples, is the limited depth of field (DOF) of the perspective views. This imposes a significant limitation on the depth range of computationally refocused images. To overcome this problem, we propose here a new method that is based on the insertion, at the pupil plane of the microscope objective, of an electrically controlled liquid lens (LL) whose optical power can be changed by simply tuning the voltage. This new apparatus has the advantage of controlling the axial position of the objective focal plane while keeping constant the essential parameters of the integral microscope, that is, the magnification, the numerical aperture and the amount of parallax. Thus, given a 3D sample, the new microscope can provide a stack of integral images with complementary depth ranges. The fusion of the set of refocused images permits to enlarge the reconstruction range, obtaining images in focus over the whole region.

This work reports the conversion of a commercial polarization microscope (Zeiss Jenaval) into an automatized Mueller matrix microscope. A Mueller matrix measurement provides the value of all the optical properties of a specimen (linear dichroism, linear birefringence, circular dichroism etc.) at a specific wavelength. In contrast to traditional polarization microscopes, which use white light and crossed polarizers to generate colored interference patterns that are analyzed by the microscope user and give only semi-quantitative results, here we demonstrate that it is possible to convert a polarization microscope into a Mueller microscope by only adding two motorized rotating compensators into the optical path without altering the optics or other opto-mechanical elements of the microscope. To our knowledge this is the most compact, fast and handy Mueller microscope ever developed. The performance of this new Mueller matrix microscope is illustrated with some birefringence measurements on textile fibers Este trabajo describe la conversión de un microscopio de polarización comercial (Zeiss Jenaval) en un microscopio de matriz de Mueller automatizado. Una medida de la matriz de Mueller proporciona todas las propiedades ópticas de una muestra (dicroísmo lineal, birrefringencia lineal, dicroísmo circular, etc) para una longitud de onda dada. Los microscopios de polarización tradicionales usan luz blanca y polarizadores cruzados para generar un patrón de colores interferencia que debe ser interpretado por el usuario del microscopio y proporciona resultados sólo semi-cuantitativos. En este trabajo demostramos que añadiendo dos compensadores rotatorios actuados por sendos motores es posible convertir un microscopio de polarización en un microscopio de matriz de Mueller sin alterar la óptica u otros elementos opto-mecánicos del microscopio. Creemos que este es el microscopio de matriz de Mueller más compacto, rápido y práctico que se ha desarrollado. El funcionamiento de este microscopio se demuestra con algunas medidas de birrefringencia en fibras textiles

Imaging Confocal Microscopes (ICM) are highly used for the assessment of three-dimensional measurement of technical surfaces. The benefit of an ICM in comparison to an interferometer is the use of high numerical aperture microscope objectives, which allows retrieving signal from high slope regions of a surface. When measuring a flat sample, such as a high-quality mirror, all ICM’s show a complex shape of low frequencies instead of a uniform flat result. Such shape, obtained from a ¿/10, Sa < 0.5 nm calibration mirror is used as a reference for being subtracted from all the measurements, according to ISO 25178-607. This is true and valid only for those surfaces that have small slopes. When measuring surfaces with varying local slopes or tilted with respect to the calibration, the flatness error calibration is no longer valid, leaving what is called the residual flatness error. In this paper we show that the residual flatness error on a reference sphere measured with a 10X can make the measurement of the radius to have up to 10% error. We analyzed the sources that generate this effect and proposed a method to correct it: we measured a tilted mirror with several angles and characterized the flatness error as a function of the distance to the optical axis, and the tilt angle. New measurements take into account such characterization by assessing the local slopes. We tested the method on calibrated reference spheres and proved to provide correct measurements. We also analyzed this behavior in Laser Scan as well on Microdisplay Scan confocal microscopes.

Non-scanning, single-shot, 3D integral microscopy with optical sectioning is presented. The method is based on the combination of Fourier-mode integral microscopy with a 3D deconvolution technique. Specifically, the refocused volume provided by a regular back-projection algorithm is 3D deconvolved with a synthetic 3D impulse response function that takes into account the number and positions of the elemental images. The use of this hybrid technique provides a stack of true-color depth-refocused images with significant gain of optical sectioning. The stack can be used, among other applications, to inspect inside the thick microscope specimen, to calculate collections of perspective views with fine angular resolution and extended full parallax, and also to display 3D images in an integral monitor. The method here presented is validated with both simulation and experimental data.

The light field microscope has the potential of recording the 3D information of biological specimens in real time with a conventional light source. To further extend the depth of field to broaden its applications, in this paper, we proposed a multifocal high-resistance liquid crystal microlens array instead of the fixed microlens array. The developed multifocal liquid crystal microlens array can provide high quality point spread function in multiple focal lengths. By adjusting the focal length of the liquid crystal microlens array sequentially, the total working range of the light field microscope can be much extended. Furthermore, in our proposed system, the intermediate image was placed in the virtual image space of the microlens array, where the condition of the lenslets numerical aperture was considerably smaller. Consequently, a thin-cell-gap liquid crystal microlens array with fast response time can be implemented for time-multiplexed scanning.

Objectives: the purpose of this study is to use light microscopy and scanning electron microscopy to determine the effect of edema on the structure of the molar vesicle. Methods: samples were taken from the complete hydatidiform mole and processed using conventional light and scanning electron microscopy techniques and an observation protocol that identified four variables: factors underlying the development of edema; the condition of the trophoblast basement membrane, development of the villi, accumulation and degeneration of sulphated mucosubstances at stromal level. Results: light microscopy showed a permeable trophoblastic basement membrane, a swollen syncytium, edematous regions disorganizating the stromal region and causing ischemic necrosis of cells. Using scanning electron microscopy, the basement membrane was found to be distended and thickened, with large irregular holes for the entry and movement of liquid, leaving a wide range of fluids during the influx process and depriving stromal cells of nutrition. Conclusions: a new three-dimensional view of the changes brought about by the entry of fluids into the stroma of molar hydropic vesicles was provided by scanning electron microscopy and confirmed by light microscopy, thereby explaining the changes occurring at the level of the stroma as an effect of the edema. Objetivos: este estudo visa utilizar a microscopia ótica e eletrônica de varredura para determinar o efeito de edema na estrutura da vesícula molar. Métodos: amostras foram obtidas da mola hidatiforme completa e processadas por técnicas de microscopia ótica convencional e eletrônica de varredura e por um protocolo de observação para identificar quatro variáveis relacionados à evolução de edema; a condição da membrana trofoblástica basal, a evolução dos vilos, a acumulação e a degeneração de mucosubstâncias sulfatadas ao nível stromal. Resultados: a microscopia ótica revelou uma membrana trofoblástica basal permeável, um sincítio inchado, regiões edematosas desorganizando a região stromal e provocando a necrose isquêmica das células. Com a microscopia eletrônica de varredura, foi observado que a membrana basal foi distendida e espessada, com grandes buracos irregulares para a entrada e movimento de líquidos, deixando uma larga variedade de fluidos durante o processo de influxo e privando as células stromais de nutrição. Conclusões: a microscopia eletrônica de varredura providenciou uma nova visualização tridimensional das alterações provocadas pela entrada de fluidos na stroma das vesículas molares, que foi verificada pela microscopia ótica, assim explicando as alterações que ocorreram ao nível da stroma como um efeito da edema.

Confocal microscopes are widely used for areal measurements thanks to its good height resolution and the capability to measure high local slopes. For the measurement of large areas while keeping few nm of system noise, it is needed to use high numerical aperture objectives, move the sample in the XY plane and stitch several fields together to cover the required surface. On cylindrical surfaces a rotational stage is used to measure fields along the round surface and stitch them in order to obtain a complete 3D measurement. The required amount of fields depends on the microscope’s magnification, as well as on the cylinder diameter. However, for small diameters, if the local shape reaches slopes not suitable for the objective under use, the active field of the camera has to be reduced, leading to an increase of the required number of fields to be measured and stitched. In this paper we show a new approach for areal measurements of cylindrical surfaces that uses a rotational stage in combination with a slit projection confocal arrangement and a highspeed camera. An unrolled confocal image of the cylinder surface is built by rotating the sample and calculating the confocal intensity in the centre of the slit using a gradient algorithm. A set of 360º confocal images can be obtained at different heights of the sample relative to the sensor and used to calculate an unrolled areal measure of the cylinder. This method has several advantages over the conventional one such as no stitching required, or reduced measurement time. In addition, the result shows less residual flatness error since the surface lies flat in the measurement direction in comparison to field measures where the highest slope regions will show field distortion and non-constant sampling. We have also studied the influence on the areal measurements of wobble and run-out introduced by the clamping mechanism and the rotational axis.

Scattering scanning near-field optical microscopy (s-SNOM) has been demonstrated as a valuable tool for mapping the optical and optoelectronic properties of materials with nanoscale resolution. Here we report experimental evidence that trapped electric charges injected by an electron beam at the surface of dielectric samples affect the sample-dipole interaction, which has direct impact on the s-SNOM image content. Nanoscale mapping of the surface trapped charge holds significant potential for the precise tailoring of the electrostatic properties of dielectric and semiconductive samples, such as hydroxyapatite, which has particular importance with respect to biomedical applications. The methodology developed here is highly relevant to semiconductor device fabrication as well.

A modified and improved model of a mechanical manipulator for observation of pinned and mounted insects is described. This device allows movement of the observed object around three perpendicular axes in the field of vision at all magnifications of stereomicroscopes. The main improvement of this new model is positioning of the guiding knobs for rotating around two of the axes next to each other, allowing faster and easier manipulation of the studied object. Thus, one of the main advantages of this device is the possibility to rotate the specimen without the need to refocus. The device enables easily reaching a precession deviation in the intersection point of axes up to 0.5 mm in the process of assembling. Um modelo modificado e melhorado de um manipulador mecânico para observação de insetos fixados e montados é descrito. Este dispositivo permite o movimento do objeto observado em torno de três eixos perpendiculares no campo de visão para todas as ampliações do microscópio. A principal melhoria deste novo modelo é o posicionamento dos botões de rotação em torno dos dois eixos ao lado do outro, permitindo uma rápida e fácil manipulação do objeto estudado. Assim, uma das principais vantagens deste dispositivo é a possibilidade de girar o espécime sem a necessidade de mudar o foco. O dispositivo permite facilmente chegar a um desvio de precisão no ponto de interseção dos eixos de até 0,5 mm no processo de montagem.

To investigate the organization of stromal collagen in healthy ex vivo corneas of different species from second harmonic generation (SHG) microscopy images.A custom backscattered nonlinear microscope has been used to study the corneal structure of different species: porcine, bovine, rabbit, rat, chicken, and humans. The instrument uses a femtosecond laser for illumination, a scanning unit, and a photon-counting detection device. It also includes a wavefront aberration control module. SHG signals produced by collagen within the cornea were acquired. A motorized stage allowed optical sectioning across the entire corneal thickness. Samples were neither fixed nor stained, and they were fully scanned.SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images could be analyzed. The anterior stroma was similar in all samples, showing interwoven short bands of collagen randomly distributed. The lamellae at the central and posterior stroma were densely packed and often presented longer bundles lying predominantly parallel to the corneal surface with characteristic spatial distributions for each species. In particular, collagen bundles in bovine and porcine corneas were interweaved. In the chick cornea, the stromal arrangement had an orientation changing regularly with depth. In human corneas, lamellae were longer and had similar orientation than their neighbors.Using a unique wavefront aberration-controlled backscattered nonlinear microscope, changes in corneal morphology as a function of depth were characterized for different species (including humans). This allowed a direct comparison among species, which might help to establish the basis of collagen distribution in animal models or to understand diseased corneas.

Digital holographic (DH) microscopy has grown into a powerful nondestructive technique for the real-time study of living cells including dynamic membrane changes and cell fluctuations in nanometer and sub-nanometer scales. The conventional DH microscopy configurations require a separately generated coherent reference wave that results in a low phase stability and a necessity to precisely adjust the intensity ratio between two overlapping beams. In this work, we present a compact, simple, and very stable common-path DH microscope, employing a self-referencing configuration. The microscope is implemented by a diode laser as the source and a Fresnel biprism for splitting and recombining the beams simultaneously. In the overlapping area, linear interference fringes with high contrast are produced. The frequency of the interference pattern could be easily adjusted by displacement of the biprism along the optical axis without a decrease in fringe contrast. To evaluate the validity of the method, the spatial noise and temporal stability of the setup are compared with the common off-axis DH microscope based on a Mach-Zehnder interferometer. It is shown that the proposed technique has low mechanical noise as well as superb temporal stability with sub-nanometer precision without any external vibration isolation. The higher temporal stability improves the capabilities of the microscope for studying micro-object fluctuations, particularly in the case of biological specimens. Experimental results are presented using red blood cells and silica microspheres to demonstrate the system performance.

La identificación diferencial de Entamoeba histolytica y Entamoeba dispar es esencial para un tratamiento adecuado del paciente y con fines epidemiológicos. Para determinar la prevalencia de E. histolytica y E. dispar se estandarizó y aplicó un ensayo de PCR, utilizando oligonucleótidos específicos para cada especie. 204 muestras de heces de individuos de la comunidad de Santa Rosa de Agua (Municipio Maracaibo, Estado Zulia, Venezuela), fueron analizadas a través del examen directo con SSF (0,85%) y lugol, concentrado de formol-éter y PCR. Al examen microscópico, 42 individuos (20,58%) presentaron formas evolutivas del complejo E. histolytica/E. dispar; mientras que la técnica de PCR evidenció un total de 47 casos positivos a estas amibas; de los cuales 22 eran portadores de E. histolytica (10,78%), 16 (7,84%) de E. dispar y 9 (4,41%) presentaron infección mixta. No hubo diferencia significativa al relacionar las variables sexo y presencia de E. histolytica y/o E. dispar, ni con los grupos etarios. No existieron casos de estas amibas, en los menores de 2 años. La frecuencia observada de E. histolytica (31/204), demuestra el carácter endémico de la amibiasis en esta comunidad.

The use of an electronically tunable lens (ETL) to produce controlled phase shifts in interferometric arrangements is shown. The performance of the ETL as a phase-shifting device is experimentally validated in phase-shifting digital holographic microscopy. Quantitative phase maps of a section of the thorax of a Drosophila melanogaster fly and of human red blood cells have been obtained using our proposal. The experimental results validate the possibility of using the ETL as a reliable phase-shifter device.

Stent quality control is a critical process. Coronary stents have to be inspected 100% so no defective stent is implanted into a human body. Skilled operators currently perform the quality process control visually, and every stent could need tens of minutes to be inspected. In this paper, a novel stent optical inspection system is presented. By the combination of a high numerical aperture microscope, a triple illumination optical system, a rotational stage, and a line-scan camera, unrolled sections of the outer and inner surfaces of the stent are obtained with high resolution at high speed. We expect with this new approach to make the stent inspection task more objective and to dramatically reduce the time and the overall cost of the stent quality control process.

The crystallization behavior of lipids has important implications in industrial processing of food products, whose physical characteristics depend largely on crystallized fats. The study of the crystallization behavior and polymorphism of a pure lipid system is of great scientific importance as a means of gaining an understanding of the phenomena involved, serving as basic knowledge to help guide the addition or removal of these compounds in different raw materials. The crystallization behavior and polymorphism of pure tripalmitin (PPP) and tristearin (SSS) were investigated by Polarized Light Microscopy (PLM) and Differential Scanning Calorimetry (DSC) under different crystallization conditions. The polymorphic forms (β' and β) of PPP and SSS exhibited different morphologies depending on how they were obtained, either from α form recrystallization or from isotropic melt. Crystallization in the β form was faster in SSS than in PPP, indicating that the process occurs faster in TAGs composed of longer fatty acid chains. Both β' and β polymorphic forms were obtained from α form recrystallization, albeit with predominance of the β form.

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.

In this paper we describe a new Mueller matrix (MM) microscope that generalizes and makes quantitative the polarized light microscopy technique. In this instrument all the elements of the MU are simultaneously determined from the analysis in the frequency domain of the time-dependent intensity of the light beam at every pixel of the camera. The variations in intensity are created by the two compensators continuously rotating at different angular frequencies. A typical measurement is completed in a little over one minute and it can be applied to any visible wavelength. Some examples are presented to demonstrate the capabilities of the instrument.

The objective of this study was to investigate the effects of blending triacylglycerols (TAGs) and diacylglycerols (DAGs) on the melting and crystallization properties in a fat system. To this end, differential scanning calorimetry (DSC), X-ray diffraction (XRD) and polarized light microscopy (PLM) methods were used. Different DAGs (diolein—OO, dipalmitin—PP and distearin—SS) were added at 5% to each TAG (triolein—OOO, tripalmitin—PPP and tristearin—SSS). DSC results showed that the addition of DAGs delayed the onset of crystallization of saturated TAGs (PPP and SSS). By contrast, the addition of DAGs to unsaturated TAG (OOO) accelerated the onset of crystallization, with the appearance of an extra crystallization peak upon the addition of SS and PP. PLM results revealed that the addition of OO affected the polymorphic transition of the TAGs studied findings were consistent with DSC melting curves and XRD results.

The buckled structure of silicene leads to the possibility of new kinds of line defects that separate regions with reversed buckled phases. In the present work we show that these new grain boundaries have very low formation energies, one order of magnitude smaller than grain boundaries in graphene. These defects are stable along different orientations, and they can all be differentiated by STM images. All these defects present local dimerization between the Si atoms, with the formation of $\pi$-bonds. As a result, these defects are preferential adsorption sites when compared to the pristine region. Thus, the combination of low formation energy and higher reactivity of these defects may be cleverly used to design new nano-structures embedded in silicene., Comment: 8 pages, 8 figures

Five cases of intraocular lens (IOL) opacification in patients implanted with the Mediphacos Ioflex® IOL are described. Clinical data in each case was obtained from the patient's medical record. The five explanted IOLs underwent gross and light microscopic analysis. Selected lenses were processed for further evaluation and multiple sagittal cuts were stained with the alizarin red and von Kossa methods. Light microscope analysis confirmed the presence of deposits on and within the lens, which stained positive for calcium. The reason why calcification has occurred in these cases remains unclear, but surgeons should be aware of this potential late postoperative complication. Cinco casos de opacificação de lente intraocular (LIO) em pacientes implantados com a LIO Ioflex® da Mediphacos são descritos. Os dados clínicos dos pacientes foram obtidos a partir dos prontuários médicos. As cinco LIOs explantadas foram enviadas para análise macroscópica e sob microscopia óptica. Algumas lentes foram processadas para análise adicional e múltiplos cortes sagitais foram corados pelos métodos de vermelho de alizarina e von Kossa. A análise sob microscopia óptica confirmou a presença de depósitos na superfície e no corpo da lente, que coraram positivamente para cálcio. A razão pela qual a calcificação ocorreu nestes casos permanece obscura. Entretanto, cirurgiões devem estar atentos para esta potencial complicação tardia.

We develop an adaptive optics (AO) multiphoton microscope by incorporating a deformable mirror and a Hartmann-Shack wavefront sensor. The AO module operating in closed-loop is used to correct for the aberrations of the illumination laser beam. This increases the efficiency of the nonlinear processes in reducing tissue photodamage, improves contrast, and enhances lateral resolution in images of nonstained ocular tissues. In particular, the use of AO in the multiphoton microscope provides a better visualization of ocular structures, which are relevant in ophthalmology. This instrument might be useful to explore the possible connections between changes in ocular structures and the associated pathologies.

A multiphoton microscope incorporating a Hartmann-Shack (HS) wavefront sensor to control the ultrafast laser beam's wavefront aberrations has been developed. This instrument allowed us to investigate the impact of the laser beam aberrations on two-photon autofluorescence imaging of human retinal tissues. We demonstrated that nonlinear microscopy images are improved when laser beam aberrations are minimized by realigning the laser system cavity while wavefront controlling. Nonlinear signals from several human retinal anatomical features have been detected for the first time, without the need of fixation or staining procedures. Beyond the improved image quality, this approach reduces the required excitation power levels, minimizing the side effects of phototoxicity within the imaged sample. In particular, this may be important to study the physiology and function of the healthy and diseased retina.

PURPOSE: To study the mechanisms of pupillary block in eyes with occludable angle by ultrasound biomicroscopy. METHODS: Initially, a pilot study of 13 eyes with acute primary angle-closure without medication was executed. Ultrasound biomicroscopy measurements of the angle, posterior chamber depth and iris thickness were performed in the temporal quadrant under light and dark conditions. Afterwards, ultrasound biomicroscopy measurements of iris-lens contact distance and iris-lens angle in the temporal quadrant and central anterior chamber depht were made in 32 eyes with acute primary angle-closure or intermittent primary angle-closure without medication, under light and dark conditions before and after laser peripheral iridectomy. RESULTS: In the pilot study, a significant decrease in the angle as well as a significant increase in the iris thickness occurred when comparing light to dark conditions. Before and after laser peripheral iridectomy (second study), significant differences were found in iris-lens contact distance (P

Made available in DSpace on 2018-12-11T17:02:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-03-01 Context: Accidental root canal perforations are among the main complications of endodontic treatment. Aim: This study evaluated the influence of operating microscope (OM) in the marginal adaptation of mineral trioxide aggregate (MTA) (Angelus® ) and glass ionomer (Vitremer) inserted into cervical perforations. Materials and Methods: Perforations were made in the cervical third of the buccal wall of the root canal in mandibular incisors. Next, the teeth were divided into four groups (N = 10): MG - MTA without OM; VG - Vitremer without OM; MOMG - MTA with OM; VOMG - Vitremer with OM. The perforations were sealed according to the group and the teeth were prepared for analysis by confocal laser scanning microscope. Images of perforation region (1,024∗) were made and the gap presented by the materials was measured using the Image J program. LEXT OLS4100 three dimensional (3D) measuring laser microscope measured the volumetric misfit. Data of gap were analyzed by Kruskal-Wallis and Dunn′s tests. Analysis of variance (ANOVA) and Tukey′s tests compared the volumetric misfits. Results: The results showed lower volume and gap in the interface dentin/material in VOMG compared to the other groups (P < 0.05). Conclusion: The use of OM improved the quality of cervical perforations sealed with Vitremer, being indicated in clinical situations of iatrogenic cervical perforations. Department of Conservative Dentistry Federal University of Rio Grande Do sul, Ramiro Barcelos St. 2492 Department of Restorative Dentistry Araraquara Dental School UNESP - Paulista State University Department of Restorative Dentistry Ribeirão Preto Dental School USP - São Paulo University Department of Restorative Dentistry Araraquara Dental School UNESP - Paulista State University

Digital Holographic Microscopy (DHM) is an optical interferometric technique for not destructive testing of micro-electro-mechanical systems (MEMS). A characterization process based on a no-contact technique allows us to analyze deformations, warping, residual stress, cracks and more other defects of MEMS, without destroy them. The flexibility of this technique allows us to improve novel numerical reconstruction algorithm for the recovery of more information. The post processing of the acquired holograms allows to reduce noise, optical aberrations, defocusing. In particular, the hologram reconstruction process has been modified to obtain Extended Focus Images (EFI). In Digital holographic microscopy, the use of microscopy objectives with high magnifications, reduces the focus depth. This means that for extended object a single reconstructed image with all the details in focus is not possible to obtain. Using a multiple reconstruction process and opportune resizing algorithms a full focused reconstructed images of extended object has been obtained without any mechanical movement. In particular, the advantages of the EFI technique are unique for dynamical characterization by DHM of extended objects, where the techniques based on multiple acquisitions fail. The EFI technique has been applied to obtain a best focused reconstructed image and profile of some micromechanical systems. It is demonstrated that this new approach allows to improve the accuracy in the EFI image when compared to the previous experimental results. Focusing of zones at different quote has been obtained evidencing, shape, crack and deformation impossible to observe otherwise at the same time. Moreover, these technique of reconstruction and analysis can be advantageous in many other fields of application.

Laser welding of corneal tissue is an alternative technique to conventional suturing procedures in ophthalmic surgery. The welding effect is achieved after staining the wound with a chromophore (Indocyanine Green, shortly: ICG) and then irradiating it with a low power diode laser. We present a study on the healing process of corneal wounds using Multispectral Imaging Autofluorescence Microscopy (MIAM). This technique is based on the characterization of fluorescence arising from tissue components (autofluorescence): it is particularly useful in studying corneal tissue, because it is mainly composed of type I collagen, one of the most important endogenous fluorophores. Laser welding tests of the cornea were carried out on rabbits in which full thickness corneal cuts of about 5 mm were sutured using a diode laser emitting at 810 nm, with a power of 80 mW. Bioptic sections of rabbit corneas were examined in a follow up study of 90 days after surgery, and the results were complementary to histological analysis performed in previous studies. Autofluorescence images showed a faster healing process and a better reorganization of the architecture of stromal fibers, in comparison with conventional suturing procedures. MIAM technique can represent a new tool to study the morphology of corneal tissue, offering some real advantages with respect to standard histological analysis. In fact, it does not require any chemical manipulation of the samples, providing information on the biological structure by directly monitoring distribution and emission intensity of endogenous fluorophores.

Purpose: To assess the capacity of methylxanthines (caffeine, theophylline, theobromine and paraxanthine) to inhibit uric acid crystallization, and to evaluate their potential application in the treatment of uric acid nephrolithiasis. Materials and Methods: The ability of methylxathines to inhibit uric acid nucleation was assayed turbidimetrically. Crystal morphology and its modification due to the effect of theobromine were evaluated by scanning electron microscopy (SEM). The ability of theobromine to inhibit uric acid crystal growth on calculi fragments resulting from extracorporeal shock wave lithotripsy (ESWL) was evaluated using a flow system. Results: The turbidimetric assay showed that among the studied methylxanthines, theobromine could markedly inhibit uric acid nucleation. SEM images showed that the presence of theobromine resulted in thinner uric acid crystals. Furthermore, in a flow system theobromine blocked the regrowth of post-ESWL uric acid calculi fragments. Conclusions: Theobromine, a natural dimethylxanthine present in high amounts in cocoa, acts as an inhibitor of nucleation and crystal growth of uric acid. Therefore, theobromine may be clinically useful in the treatment of uric acid nephrolithiasis., This work was supported by project grants CTQ2010-18271/PPQ and TEC2010-19467 from the Ministerio de Ciencia e Innovacion (Gobierno de Espana), FEDER funds (European Union) and project grant 9/2011 from the Conselleria d'Educacio, Cultura i Universitat (Govern de les Illes Balears). A.R. is grateful to the European Social Fund and the Conselleria d'Educacio, Cultura i Universitats for the fellowship FPI/1570/2013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

New methods of image analysis and three-dimensional pattern recognition were developed in order to perform the automatic scan of nuclear emulsion pellicles. An optical microscope, with a motorized stage, was equipped with a CCD camera and an image digitizer, and interfaced to a personal computer. Selected software routines inspired the design of a dedicated hardware processor. Fast operation, high efficiency and accuracy were achieved. First applications to high-energy physics experiments are reported. Further improvements are in progress, based on a high-resolution fast CCD camera and on programmable digital signal processors. Applications to other research fields are envisaged.

SUMMARY The paper describes an automatic pulsed laser microfluorometer with high spatial and temporal resolution, developed in our laboratories. The instrument consists of: (i) a nitrogen-laser-pumped dye-laser for the excitation of the fluorescence, (ii) a microscope with additional optics to focus the excitation beam on the sample and to collect the fluorescence, (iii) filters or monochromators to select the output wavelength, (iv) a fast photomultiplier tube to detect the signal, and (v) a dual time-scale microprocessor-controlled signal averager for the acquisition and processing of the signal. Examples are given that show the potential of the time-resolved fluorescence microscopy in studying, quantitatively and qualitatively, the properties of fluorescent molecules.

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view. This work is supported by the Generalitat de Catalunya grant 2009-SGR-159, the Spanish government grants TEC2009-09698 and SAF2008-00211, the EU project STELUM (FP7-PEOPLE-2007-3-1-IAPP, 217997), the Swiss National Science Foundation (SNSF) Sinergia project grant CRSII3_125447, the FP7-NMP VIBRANT project grant CP-IP 228933-2, the NoE P4L and Laserlab optobio. Jacob Licea-Rodriguez acknowledges CONACYT Mexico for supporting his stay at ICFO through a mixed fellowship. This research has been partially supported by Fundació Cellex Barcelona and was mainly conducted at ICFO’s “Super-Resolution Light Microscopy and Nanoscopy Facility”

Super-resolution microscopy is increasingly becoming an important tool for biological research, providing valuable information at the nanometer length scales inside cells and tissues. In the past decade numerous technological advancements have transformed superresolution microscopes into powerful tools of discovery. While the first super-resolution images took several hours to acquire, recent progress has led to tremendous improvement in acquisition speed, enabling researchers to probe dynamic processes in living cells with unprecedented spatiotemporal resolution. This mini-review focuses on the recent developments in live-cell super-resolution microscopy and its biological applications. This work was supported in part by Fundació Cellex, Barcelona, Marie-Curie International Reintegration Grant FP7-PEOPLE-2010-RG and the Plan Nacional Grant from the Spanish Ministry (Ministerio de Economia y Competitividad) FIS2012-37753.

Light field technologies have seen a rise in recent years and microscopy is a field where such technology has had a deep impact. The possibility to provide spatial and angular information at the same time and in a single shot brings several advantages and allows for new applications. A common goal in these applications is the calculation of a depth map to reconstruct the three-dimensional geometry of the scene. Many approaches are applicable, but most of them cannot achieve high accuracy because of the nature of such images: biological samples are usually poor in features and do not exhibit sharp colors like natural scene. Due to such conditions, standard approaches result in noisy depth maps. In this work, a robust approach is proposed where accurate depth maps can be produced exploiting the information recorded in the light field, in particular, images produced with Fourier integral Microscope. The proposed approach can be divided into three main parts. Initially, it creates two cost volumes using different focal cues, namely correspondences and defocus. Secondly, it applies filtering methods that exploit multi-scale and super-pixels cost aggregation to reduce noise and enhance the accuracy. Finally, it merges the two cost volumes and extracts a depth map through multi-label optimization.