A large decrease in the side effects of a drug can be obtained if it is efficiently delivered in a timely manner and in the needed location only. By combining therapeutic and diagnostic (theranostic) functionalities with targeting capabilities and large surface areas, nanoparticles provide an ideal vehicle for personalized medicine.

The main objective of our work is to develop hybrid Mesoporous Silica Nanoparticles (MSNs) for theranostics, carrying fluorescent beacons for traceability and imaging, featuring a smart release control mechanism, able to accommodate large drug loads and to deliver their cargo on demand to a desired location. This communication focus on the preparation of the fluorescent MSNs, and characterization by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and laser scanning fluorescence confocal microscopy (LSFCM).

Mesoporous Silica Nanoparticles (MSNs) with well-defined and controllable particle morphology are exceptional supports/nanocontainers for molecules and polymers.This class of materials are characterized by an ordered pore system of 2-8nm diameter, pore volumes above 1mL/g and particle size from 40nm to several hundred nanometers. The preparation of fluorescent hybrid MSNs involves the presence of a fluorescent molecule during particle synthesis, which becomes aligned with the pores, thus impervious to aggregation and self-quenching effects. The MSNs external surface can be selectively functionalized to immobilize polymers or (bio)molecules for possible targeting or sensing, and the pore is available for solvent diffusion, allowing the incorporation of different molecules.

We prepared monodispersed hybrid MSNs incorporating a fluorescent perylenediimide (PDI) derivative in the wall structure. The MSN-PDI were characterized by TEM (Figure 1), SEM and fluorescence emission spectroscopy. LSFCM images of the MSN-PDI after incubation in HEK293 cells show the internalization of the nanoparticles (Figure 2). These new hybrid nanoparticles, after surface-functionalization with stimuli-responsive gate systems, open possibilities for the development of traceable drug delivery systems.

This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTM/101627/2008 and PEst-OE/CTM/LA0024/2011. T.R. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009) and Pos-Doc (SFRH/BPD/64320/2009) grants.

The detailed knowledge of the three-dimensional (3D) organization of the nervous tissue provides essential information on its functional elucidation. We used serial block-face scanning electron microscopy with focused ion beam (FIB) milling to reveal 3D morphologies of the mossy fiber rosettes in the mice cerebellum. Three-week-old C57 black mice were perfused with a fixative of 1% paraformaldehyde/1% glutaraldehyde in phosphate buffer; the cerebellum was osmicated and embedded in the Araldite. The block containing granule cell layer was sliced with FIB and observed by field-emission scanning electron microscopy. The contrast of backscattered electron image of the block-face was similar to that of transmission electron microscopy and processed using 3D visualization software for further analysis. The mossy fiber rosettes on each image were segmented and rendered to visualize the 3D model. The complete 3D characters of the mossy fiber rosette could be browsed on the A-Works, in-house software, and some preliminary quantitative data on synapse of the rosette could be extracted from these models. Thanks to the development of two-beam imaging and optimized software, we could get 3D information on cerebellar mossy fiber rosettes with ease and speedily, which would be an additive choice to explore 3D structures of the nervous systems and their networks.

Mathematical morphology is a novel geometry-based technique for image processing and analysis, originally developed to process binary images, based on the use of simple concepts from set theory and geometry such as set inclusion, intersection, union, complementation, and translation Geometry is used to represent object shape, physics inflict constraints on how the shape may vary over space and time, and optimal approximation theory make available the formal underpinnings of mechanisms for fitting the models to measured data. Deformable curve, surface, and solid models gained popularity after they were proposed by Terzopoulos for use in computer vision and computer graphics in the mid 1980’s introducing the theory of continuous (multidimensional) deformable models in a Lagrangian dynamics setting, based on deformation energies in the form of generalized splines. The deformable model that has attracted the most attention to date is popularly known as “snakes”, planar deformable contours that are useful in several image analysis tasks. They are often used to approximate the locations and shapes of object boundaries in images based on the reasonable assumption that boundaries are piecewise continuous or smooth.

This work presents a novel approach since it joins (and, indeed, reinforces) the index framework with the evaluation of the same biological samples by a suitable combination of deformable models. Nucleus contour is identified through Active Shape Models techniques, and cytoplasm contour’s detected through parametric Snakes, with prior image preprocessing based on statistical and mathematical morphology techniques. Morphometric parameters such as nucleus and cytoplasm area and ratio between them are then easily computed. Biometry was performed using an ocular micrometer and nucleus/cytoplasm ratios were obtained characterizing each of the three identified stages: Immature, Vitellogenic and Mature. This resulted in a collection of tools, called morphological operators, which are eminently suited for the analysis of shape and structure in binary images. Acartia tonsa was used as model to establish an index for oocyte maturity determination based in citometry and histochemical evaluation of gonadic masses. It was validated the application of a methodology with a realistic background and a new, more accurate and ecologically realistic index for oocyte staging emerged.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Leaf stomatal characteristics of Siberian elm (Ulmus pumila) were investigated by electron microscopy and white light scanning interferometry. On the basis of average annual precipitations, two types of tree specimens were collected from Korea, China, and Mongolia: (1) trees under normal environmental conditions and (2) trees under arid conditions. Field emission scanning electron microscopy revealed oval-shaped stomata on the lower surface, and they were ca. 20 μm in width. In-lens secondary electron imaging showed differences in electron density and stomatal pore depth between the two types. According to the line profile analysis by white light scanning interferometry, stomata under arid conditions appeared to have higher levels of the stomatal pore depth than ones under normal conditions. Focused ion beam–field emission electron microscopy supported the increased stomatal pore depth with the increasing drought stress gradient. These results suggest that complementary microscopy can be employed to unravel the adaptive phenotypic plasticity of Siberian elm in response to drought stress.

Species of Aspergillus from section Nigri, also known as black aspergilli, are distributed worldwide and have been widely used for purposes of various types such as: biotechnological, industrial, medical, among others. They have been extensively studied for being the causing agents of biodeterioration of commodities and food.

Within this section, new species have been recently described and among them Aspergillus ibericus and Aspergillus uvarum were both isolated from grapes. The polyphasic approach used in the analysis of these species, either by morphological techniques as well as by molecular methods, allowed not only their characterisation but also their consequent separation from all others in the section. Microbial taxonomy has the identification of species as one of its fundamental goals. Data such as: morphological characteristics description, physiological and biochemical properties, ecological roles, and societal risks or benefits, are key elements in any fungal identification process. But, this process is subjected to periodic changes due to frequent revisions of the taxonomic schemes, therefore becoming time consuming and more demanding and difficult, even for skilled specialists. Furthermore, each taxonomic group has specialised literature, terminology and characters. This takes place since identifications have difficulties of consensual naming, depending on the criteria used and the amount of information available when producing all data. It is increasingly becoming clear that, to better achieve a more accurate concept of species, microbial identification and authentication require a polyphasic approach to produce consistent, useful and quality data.

Characterisation of morphologic and structural aspects of spores from Aspergillus strains, section Nigri, has been carried out using scanning electron microscopy (SEM) and intends to contribute to the associated data of the strains from this section.

Colonies from 13 Aspergillus from section Nigri, from the Micoteca da Universidade do Minho (MUM), were grown at 25 ºC for 3 or 4 days in malt extract agar directly mounted in a sterilised SEM stub. The samples were covered with a mixture of gold and platinum (80/20%) and then examined in the SEM [NanoSEM - FEI NovaTM 200 (FEG/SEM); EDAX - Pegasus X4M (EDS/EBSD)]. A stereomicroscope (Leica MZ12.5) was used to have a clear image comparison of the size of the conidial head.

Although the analysed strains presented dimensional, morphological and structural diversity, common or typical features could be inferred and related to each taxon like those represented in Fig 1.

From the SEM analysis we were able to conclude that in the section Nigri of genus Aspergillus the spore wall ornamentation, and its size and shape continue to present themselves as important primary diagnostic traits for species differentiation.

Electron tomography (ET) has recently afforded new insights into neuronal architecture. However, the tedious process of sample preparation, image acquisition, alignment, back projection, and additional segmentation process of ET repels beginners. We have tried Hitachi's commercial packages integrated with a Hitachi H-7650 TEM to examine the potential of using an automated fiducial-less approach for our own neuroscience research. Semi-thick sections (200–300 nm) were cut from blocks of fixed mouse (C57BL) cerebellum and prepared for ET. Sets of images were collected automatically as each section was tilted by 2° increments (±60°). “Virtual” image volumes were computationally reconstructed in three dimension (3D) with the EMIP software using either the commonly used “weighted back-projection” (WBP) method or “topography-based reconstruction” (TBR) algorithm for comparison. Computed tomograms using the TBR were more precisely reconstructed compared with the WBP method. Following reconstruction, the image volumes were imported into the 3D editing software A-View and segmented according to synaptic organization. The detailed synaptic components were revealed by very thin virtual image slices; 3D models of synapse structure could be constructed efficiently. Overall, this simplified system provided us with a graspable tool for pursuing ET studies in neuroscience.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Paget Disease of Bone is a disorder characterized by abnormal bone remodeling, associated with morphological and functional abnormalities of osteoclasts although its etiology remains unproven. Ion beam analysis (IBA) techniques may be useful to characterize the abnormal distribution and concentration of the major and trace elements that can be associated topagetic bone condition. In order to access the viability of this approach, healthy femoral head of cat has been investigated by Particle Induced X-Ray Emission (PIXE),Rutherford Backscattering Spectrometry (RBS) and Scanning Electron Microscopy (SEM).

The bone section was irradiated, under vacuum conditions, with a 2.0 MeV alpha particle beam produced by the 2.5 MV Van de Graaff Accelerator of CTN/IST (Lisbon, Portugal). An Oxford Microbeams-type nuclear microprobe was used (OM150 triplet system), which allowed the proton beam to be focused on the bone section with a spatial resolution of 3x4 µm2 and a mean beam current of ~90pA. The X-rays generated by the bone were recorded using an 80 mm2 Link X-ray detector (with a 145 eV energy resolution), that is positioned at 135º to the beam direction. The backscattered particles were collected by a 200 mm2 silicon surface barrier radiation detector with a 15 keV energy resolution that is positioned at 40º to the beam direction in a cornell geometry. SEM observations have been carried out with backscattered electrons (BSE) using a JEOL JSM 7001F microscope equipped with an INCA Oxford Instruments EDS spectrometer for point analyses and X-ray mapping. Standard petrographic procedures have been employed to prepare the samples for SEM observation: the specimens were partly encapsulated in epoxy resin, ground with 800 and 1000-grit SiC paper, polished with 6 μm and 1 μm diamond suspensions and coated with carbon.

A selected area of the sample has been scanned with the proton beam, allowing to generateelemental distribution maps for Ca and P (Figure 1). From X-line scans it was possible to extractline profiles of these elements. RBS and PIXE were used to determine the matrix and trace element composition in order to normalize the elemental concentrations. The results are shown in Table 1.Structural information on the Haversian Systems, includingbone lacunaeandosteocytes localization have been obtained by SEM (Figure 2).

The combination of these techniques proved to be a viable approach for the Pagetic bone characterization and will be used to analyze the structure of trabecular and cortical bones as well as the distribution and morphology of the pagetic bone cells, which can contribute to clarify the differences between pagetic and healthy bone.

V. Corregidor acknowledges the support from FCT-Ciência program. The work was also supported by FCT PEST-OE/CTM-UI0084/2011 grant.

At the apical tip of Drosophila testis, there is a stem cell niche known as the proliferation center, where the stem cells are maintained by hub cell cluster for the regulation of differentiation and proliferation. Germline stem cells go through mitosis four times from one primary spermatogonial cell to the 16-cell stage before the maturation. The cells derived from the same germline stem cell are located within one cyst, an enclosed system by two cyst cells, and they are connected by the intercellular bridges called ring canals. In this study, the three-dimensional (3D) structure of Drosophila testis tip was reconstructed from serial sections. The size of cells at each stage was compared in volume from the 3D structure. The stages of cells in a cyst could be distinguishable exactly by counting the cells linked with intercellular bridges in 3D-reconstructed structure. The cysts containing the same stage cells appeared in the horizontal plane. Both the germline stem cell directly attached to the hub cell and the spermatogonial cells detached from the hub cell were divided at the almost perpendicular direction to the spermatogonial cell layers. The dividing phase in one cyst was delayed gradually through the cytoplasmic region of intercellular bridge.

Laser scanning fluorescence confocal microscope (LSFCM) imaging is an extensively used modality in biological research. However, these images present low signal to noise ratio and a time intensity decay effect due to the so called photoblinking/photobleaching (PBPB) phenomenon that corresponds to an intensity fading of a fluorescent probe along the time, as shown in Figure 1. This effect is caused by quantum phenomena associated with the electronic excitation and photochemical reactions among the fluorescent and the surrounding molecules induced by the incident radiation that temporarily or irreversibly destroy their ability to fluoresce. Since illumination is needed to excite and observe the tagging fluorescent proteins in the specimen and all the fluorophores will eventually photobleach upon extended excitation, the acquisition of this type of images becomes a hard task for long exposures.

There are in the literature several proposals to model this fading effects and among them the single and double exponential are the most used. However, simple and tractable theoretical models based on the physics of the observation process to support these empirical laws are not available. In this work, that theoretical model, supported on the underlying physics of the process, is derived to describe the PBPB effect (see Figure 2).

From a fluorescence point of view, tagging molecules can be in three main states (see Figure 3), i) ON-state, where they are able to fluoresce and be observed, ii) OFF-state, where they are temporarily not able to fluoresce and therefore are not visible and finally at the iii) BLEACHED-state where they become permanently OFF. Here, a continuous time differential equation dynamic model is proposed to describe the number of molecules at the ON-state, nON, along the time. The model is based on the underlying quantum mechanic physics theory of the observation process associated with this type of images and the common empirical weighted sum of two decaying exponentials (DExp), usually used in the literature, is derived from the model. The parameters βON and βOff are the transitions rate from and to the ON-state respectively and ξis the decay rate associated with the transitions for the permanent BLEACHED state.

Experiments with synthetic and real data are presented to validate the PBPB model and estimate the physical variables associated with the process. Intensity decay from real data and the corresponding theoretical curve are compared and displayed in Figure 4.

This work was supported by the FCT project [PEst-OE/EEI/LA0009/2011]. The authors thank Dr. José Rino and Profa Carmo Fonseca, from the Molecular Medicine Institute (IMM) of Lisbon, for providing biological technical support and the real data used in this paper.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The increase in CO2 emissions from anthropogenic sources may not only result in temperature increase on a global scale but also in increased ocean acidification (OA), by lowering the ocean's capacity to absorb additional atmospheric CO2. As a consequence, changes in ocean chemistry are prone to occur through the imbalancing of sea-atmosphere gas exchange, thus affecting O2 absorption as well.

There are many studies on the potential effects of OA and increased ocean temperatures on the physiology of marine organisms but little is known about changes at histological level and there are still several gaps at molecular level which must be studied for a better understanding of all biological mechanisms involved. With regard to hypoxia, alterations to the stress response can provide information on the organisms’ physiological effects and coping strategies triggered by anoxia. For instance, it is known that organisms respond by reducing protein synthesis. Once temperature affects physiological, behavioral and ecological processes, there is a need to understand what mechanisms are behind the organisms’ response to stress, enhancing our predictive and environmental management capacities considering a climate change scenario. This is of great importance, in particular to countries with a sea-based economy.

The aim of the present study is to assess the stress response of a marine fish, sea bream (Sparus aurata) exposed to increasing water temperature and different water acidity (alone or in combination). Here we present preliminary data on temperature effects on S. aurata at a cellular and molecular level. In addition, tissue samples from muscle, livers, gills and intestine are examined to evaluate any alterations caused by altering this physical parameter.

Fish were distributed randomly in tanks (n=96) and allowed to acclimate at 18ºC (the same temperature of the hatchery) before the beginning of the bioassays. After assessment of the upper thermal limits (UPL), water temperature was increased at a rate of 1ºC per hour using a thermostatized bath with a constant rate of water-temperature until reaching the endpoint, following the dynamic method of Critical Thermal Maximum (CTM). Every 2ºC step, fish were euthanized by cervical transection and the selected organs removed and stored at -80ºC until further analysis. Sub-samples were taken and processed for histological examination following standard techniques. Frozen samples were analysed for heat stress proteins (HSP70) as described by Madeira et al. The histological observations were carried out using a Leica microscope (DMLB model). Preliminary results of the histological examination showed changes in the cellular structure, with visible damage at higher temperatures in liver and gills (Fig. 1). Regarding HSP70, significant changes were observed throughout the temperature assay. The results indicate that elevated water temperature can be a major stressor that will affect fish due to potential climate changes, thus compelling the need to perform these studies to enhance our predictive and environmental management capacities.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/MAR/119068/2010 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The “LEXT” confocal laser scanning microscope has been used for the three-dimensional (3D) imaging of the surface of specimens, especially in materials science fields, by the penetration of near-infrared (NIR) light without mechanical cutting, deposition, or other specimen pretreatment. Noninvasive investigation of various biological tissues such as human spinal dura mater, rat aorta, and cornea without the dehydration process was successfully carried out with the “LEXT,” in order to access both surface and internal topographic images of the biological structures at a good status of the wet tissue such as in vivo, especially in measuring tissue thickness. The confocal NIR laser microscopy offers the viable means to visualize tissue architecture and its thickness in microdomain to integrate 3D images efficiently. We believe that the “LEXT” has a good application for biological researchers to study biomaterials, and it would be useful as a diagnostic tool in the near future.