New advances in scanning microscopy and its application to study parasitic protozoa.

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities.

Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development.

Preparación de una matriz extracelular tridimensional por descelularización de hígados de conejos / Preparación de una matriz extracelular tridimensional por descelularización de hígados de conejos / Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

Introducción: el número de hígados trasplantables es insuficiente para satisfacer las necesidades actuales de la demanda de injerto. La búsqueda de alternativas terapéuticas ha generado diferentes líneas de investigación, una de ellas es la utilización de matrices biológicas tridimensionales descelularizadas y la posterior siembra celular para obtener un órgano funcional. Objetivo: obtención de un protocolo de descelularización de hígado de conejo que genere una matriz hepática tridimensional. Métodos: una combinación de detergentes (Triton X-100 y SDS), agentes físicos y enzimáticos se utilizaron para descelularizar hígados de conejo. Los órganos se pefundieron en forma retrógrada con distintos agentes químicos durante 68 horas. Luego los hígados se examinaron por técnicas morfológicas (microscopía óptica y electrónicade barrido) y bioquímicas (cuantificación de ADN) para evaluar una correcta descelularización así como la obtención de una matriz extracelular preservada. Resultados: la observación macroscópica del órgano permitió inferir la descelularización del mismo. Las tinciones macroscópicas utilizadas mostraron una correcta conservación de los árboles biliar y vascular. Por otro lado, la observación microscópica del hígado permitió corroborar los resultados macroscópicos observados, la tinción de hematoxilina-eosina mostró ausencia de células y de material nuclear así como la presencia de la tríada portal. La tinción de Wilde evidenció la conservación de las fibras de reticulina en la matriz descelularizada. Asimismo, la microscopía electrónica de barrido reveló una cápsula de Glisson conservada y la descelularización de la cuantificación de ADN fue inferior al 10 % en el hígado descelularizado con respecto al hígado control. Finalmente, el tiempo utilizado para la descelularización fue inferior a las 96 horas. Conclusiones: el protocolo de descelularización propuesto fue apropiado ya que se verificó una ausencia de células y una matriz hepática con conductos vasculobiliares conservados y con una arquitectura tridimensional adecuada para una futura siembra celular(AU)

Introduction: the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. Objective: to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. Methods: a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. Results: the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin- eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde’s staining demonstrated the conservation of reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson’s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. Conclusions: the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding(AU)

Partículas de polietileno en líquido sinovial tras artroplastia de rodilla con un polietileno convencional o uno altamente entrecruzado. Estudio preliminar / Polyethylene particles in synovial fluid after knee arthroplasty with a conventional or highly cross-linked polyethylene. Preliminary study

Objetivo. En los últimos años han aparecido polietilenos altamente entrecruzados para intentar disminuir el desgaste, tal como se ha demostrado en simuladores de rodilla. El objetivo es evaluar mediante el recuento de partículas de polietileno en líquido sinovial, si se confirma un menor desgaste en pacientes con prótesis de polietileno altamente entrecruzado. Material y método. Estudio prospectivo aleatorizado, en el que durante la implantación de una prótesis de rodilla se asignó a un grupo de pacientes la colocación de un polietileno convencional (grupo A), y a otro grupo un polietileno altamente entrecruzado (X3® de Stryker Orthopaedics) (grupo B). A los 12 meses tras la cirugía se practicó una artrocentesis de rodilla y se hizo un recuento de partículas de polietileno en el líquido sinovial mediante microscopio electrónico de barrido. Se han analizado 14 muestras en cada grupo. Resultados. Ambos grupos son comparables en todas las variables estudiadas. No hemos hallado diferencias significativas en la concentración de partículas de polietileno/ml (1,49±0,85 millones grupo A vs. 1,42±0,91 millones grupo B; p=0,60) ni en el número total de partículas aisladas entre ambos grupos. Tampoco hemos hallado diferencias en el tamaño ni la morfología de partículas entre ambos grupos. Discusión y conclusiones. Aunque diversos trabajos in vitro han hallado una reducción muy significativa del desgaste del polietileno altamente entrecruzado, no se ha hallado que en las prótesis de rodilla implantadas en pacientes este desgaste se vea reducido. La gran variabilidad del número de partículas entre individuos sugiere que el desgaste de polietileno in vivo depende de muchos factores y probablemente el tipo de polietileno no sea el más determinante (AU)

Aim of the study. In recent years cross-linked polyethylenes have been developed in an attempt to reduce the wear, as has been demonstrated in knee simulators. The aim is to assess, by counting particles of polyethylene in synovial fluid, whether the reduction in wear is confirmed in patients with a highly crosslinked polyethylene prosthesis. Material and methods. A prospective randomised study was designed. During the implantation of a knee prosthesis, one group of patients was assigned the use of a conventional polyethylene (group A), and the other group a highly crosslinked polyethylene (X3®, Stryker Orthopaedics) (group B). At 12 months after surgery a knee arthrocentesis was performed, and the number of polyethylene particles was counted in a scanning electron microscopy. Fourteen samples from each group were studied. Results. Both groups were comparable in all study variables. We found no significant differences in the concentration of polyethylene particles/ml (1.49±0.85 million in group A vs 1.42±0.91 million in group B, P=.60) or the total number of isolated particles. We found no differences either in size or morphology of particles between both groups. Discussion and conclusions. Although several in vitro studies in vitro using different types of highly crosslinked polyethylene found a significant reduction, we did not find that that wear was reduced in the knees of these patients. The great variability in the number of particles between individuals suggests that polyethylene wear in vivo depends on many factors, so perhaps the type of polyethylene is not the most significant factor (AU)

Effect of surfactant on the release of ciprofloxacin from gelatin microspheres

Gelatin microspheres with high entrapment efficiency were prepared by emulsification (using differentsurfactants) and crosslinking by glutaraldehyde with a view to improve stability of the microspheres.The microspheres were characterized for entrapment efficiency of ciprofloxacin, swelling, FT-IR,scanning electron microscopy (SEM) and leakage properties.From the results obtained we inferred thatthe gelatin microspheres entrapped with ciprofloxacin prepared by using three different surfactantsincreased the shelf life of gelatin microspheres(AU)

Método de preparación del espécimen para evaluar la micromorfología de la interfase adhesiva resina-dentina con un microscopio electrónico de barrido / No disponible

La infiltración con resina de las fibras de colágeno desmineralizadas permiten la formación de una capa híbrida con tags de resina y ramas laterales, creando de esta manera, retenciones micromecánicas de la resina al substrato desmineralizado. Dichas retenciones son el principal mecanismo para la adhesión resina-dentina, siendo la penetración de la resina en la dentina intertubular, el factor de mayor importancia en la obtención de unas adecuadas fuerzas de adhesión. Uno de los instrumentos más usados para estudiar los mecanismos que envuelven el proceso de adhesión ha sido el microscopio electrónico de barrido (MEB). El principio del MEB se basa en imágenes tridimensionales que se construyen punto a punto y línea a línea desde los electrones secundarios. El objetivo de este estudio es describir la preparación de los espécimenes para evaluar la formación de la capa híbrida, los tags de resina y ramas laterales (AU)

The infiltration of demineralized collagen fibers with resin permits formation of a hybrid layer with resin tags and adhesive lateral branches, thus creating micromechanical retention of the resin to the demineralized substrate. The micromechanical retention is the chief mechanism for resin bonding to dentin with resin penetration of the intertubular dentin being of major importance in bond strength. One of the first and most widely used tools to study the mechanisms involved in the process of bonding has been the scanning electron microscope (SEM). The principle of SEM is based upon a pseudo three-dimensional image that is built up point-by-point and line-by-line from secondary electrons. The objective of this study is to describe the preparation of the specimens to evaluate the hybrid layer, resin tags and adhesive lateral branches formations (AU)

A history of scanning electron microscopy developments: towards "wet-STEM" imaging.

A recently developed imaging mode called "wet-STEM" and new developments in environmental scanning electron microscopy (ESEM) allows the observation of nano-objects suspended in a liquid phase, with a few manometers resolution and a good signal to noise ratio. The idea behind this technique is simply to perform STEM-in-SEM, that is SEM in transmission mode, in an environmental SEM. The purpose of the present contribution is to highlight the main advances that contributed to development of the wet-STEM technique. Although simple in principle, the wet-STEM imaging mode would have been limited before high brightness electron sources became available, and needed some progresses and improvements in ESEM. This new technique extends the scope of SEM as a high-resolution microscope, relatively cheap and widely available imaging tool, for a wider variety of samples.

Scanning electron microscopy of cell surface morphology.

The surface of metazoan cells is a landscape not clearly visualized by light microscopy. Many cells elaborate protrusive structures such as microvilli, filopodia, lamellipodia, and surface ruffles that play important roles in the interaction between the cell and its environment. The high resolution of scanning electron microscopy makes it an ideal technique for studies of the cell surface; however, preservation of fine surface structure can be problematic. Here we highlight the critical factors in sample preparation to ensure optimal high-resolution imaging of the surface of mammalian cells and tissues.

Chemically imaging living cells by scanning electrochemical microscopy.

Scanning electrochemical microscopy (SECM) is useful in probing and characterizing interfaces at high resolution. In this paper, the general principles of this technique are described and several applications of SECM to biological systems, particularly to living cells, is discussed, along with several example systems. Thiodione was detected and monitored electrochemically during the treatment of hepatocytes with cytotoxic menadione. The antimicrobial effects of silver(I) was followed by SECM through bacterial respiration. Living HeLa cells were shown to accumulate ferrocencemethanol (FcMeOH) and generated positive feedback for FcMeOH oxidation that can be further used to monitor the cell viability. Finally, individual giant liposomes, as cell models, with encapsulated redox compounds were successfully probed by SECM. In general SECM has the advantage of very high spatial resolution and versatility, especially for the detection of electroactive substances.

Recent developments and new strategies in scanning electron microscopy.

In addition to improvements in lateral resolution in scanning electron microscopy, recent developments of interest here concern extension of the incident beam energy, E(0), over two decades, from approximately 20 keV to approximately 0.1-0.5 keV and the possibility of changing the take-off emission, alpha, of detected secondary electrons. These two degrees of freedom for image acquisition permit a series of images of the same field of view of a specimen to be obtained, each image of the series differing from the others in some aspect. The origins of these differences are explored in detail and they are tentatively interpreted in terms of the change in the secondary electron emission yield delta vs. E(0), delta = f(E(0)), and also of the change in delta vs. alpha, partial differentialdelta/ partial differentialalpha. Various origins for the chemical contrast and topographic contrast have been identified. Illustrated by correlating a secondary electron image and a backscattered electron image, use of the scatter diagram technique facilitates image comparison. The difference between the lateral resolution and the size of the minimum detectable detail is outlined to avoid possible errors in nanometrology. Some aspects related to charging are also considered and possible causes of contrast reversal are suggested. Finally, the suggested strategy consists of the acquisition of various images of a given specimen by changing one parameter: primary beam energy and take-off angle for conductive specimens; working distance or beam intensity for high-resolution experiments; scanning frequency for insulating specimens.

Static and dynamic charges: changing perspectives and aims in electron microscopy.

In the context of electron microscopists' changing attitudes to charging effects, some basic aspects of these phenomenona are surveyed. Methods of mapping internal charge distributions such as doping levels in semiconductors, trap distributions, or internal electric fields in insulators are discussed.

Recent advances in electron imaging, image interpretation and applications: environmental scanning electron microscopy.

One of the latest developments in electron microscopy is the environmental scanning electron microscope (ESEM), which enables soft, moist and/or electrically insulating materials to be viewed without pre-treatment, unlike conventional scanning electron microscopy, in which specimens must be solid, dry and usually electrically conductive. Such an advance has significant implications for studies of the 'native' surfaces of specimens including rocks and minerals, polymers, biological tissues and cells, food and pharmaceutical products, precious artefacts and forensic material, for example. Previous types of electron microscopes made scientists think carefully about the physics of electron-beam interactions with specimens and, hence, the interpretation of images. We now face additional factors influencing the emission and detection of electron signals, unique to the imaging of specimens in the partial vacuum of an ESEM. Just as importantly, we must consider the thermodynamic and kinetic stability of specimens, as appropriate, and explore the possibilities for new applications, particularly those of a dynamic nature. This paper briefly describes some of the issues involved and reviews the current state of understanding.

The use of environmental scanning electron microscopy for imaging wet and insulating materials.

The environmental scanning electron microscope (ESEM) is a direct descendant of the conventional SEM, but also permits wet and insulating samples to be imaged without prior specimen preparation. A low pressure (up to around 10 torr) of a gas can be accommodated around the sample. When this gas is water, hydrated samples can be maintained in their native state. Whether the gas is water or some other gas, ions formed on collisions between electrons emitted from the sample and the gaseous molecules drift back towards the sample surface helping to reduce charge build up. This eliminates the need for insulators to be subjected to a conductive surface coating. These two key advantages of ESEM open up a wide range of materials to the power of scanning electron microscopy.

The development of optical nanosensors for biological measurements.

This article discusses and documents the basic concepts of, and developments in, the field of optical nanosensors and nanobiosensors. It describes the progression of this field of research from its birth up to the present, with emphasis on the techniques of sensor construction and their application to biological systems. After a brief overview of the techniques for fabricating nanometer-sized optical fibers, we describe the various types of transducer and bioreceptor molecule presently used for nanosensor and nanobiosensor fabrication.

Double-layer coating for field-emission cryo-scanning electron microscopy--present state and applications.

Imaging of fast-frozen samples is the most direct approach for electron microscopy of organic material. It prevents chemical fixation and drying artifacts. Frozen samples can be replicated and imaged in the transmission electron microscope (TEM), or they can be directly visualized in the cryo-scanning electron microscope (cryo-SEM). Double-layer coating combines these two techniques and many of their advantages. With this method, the frozen bulk sample is coated similar to the TEM-replica technique with, for example, a shadow of platinum (at an angle of 45 degrees) and an additional layer of carbon. Then, the sample is cryo-transferred to an SEM equipped with a cold stage and imaged with the material-dependent backscattered electron signal that shows the platinum distribution. With this method, charging artifacts and the effects of beam damage are significantly reduced. Although currently the resolution of the replica technique cannot be surpassed, the method greatly facilitates the processing of brittle, rapidly frozen samples because no replica cleaning is necessary. This makes the method especially suitable for high-pressure frozen samples.