Trajectories of individual molecules moving within complex environments such as cell cytoplasm and membranes or semiflexible polymer networks provide invaluable information on the organization and dynamics of these systems. However, when such trajectories are obtained from a sequence of microscopy images, they can be distorted due to the fact that the tracked molecule exhibits appreciable directed motion during the single-frame acquisition. We propose a new model of image formation for mobile molecules that takes the linear in-frame motion into account and develop an algorithm based on the maximum likelihood approach for retrieving the position and velocity of the molecules from single-frame data. The position and velocity information obtained from individual frames are further fed into a Kalman filter for interframe tracking of molecules that allows prediction of the trajectory of the molecule and further improves the precision of the position and velocity estimates. We evaluate the performance of our algorithm by calculations of the Cramer-Rao Lower Bound, simulations, and model experiments with a piezo-stage. We demonstrate tracking of molecules moving as fast as 7 pixels/frame (12.6 μm/s) within a mean error of 0.42 pixel (37.43 nm).

Metabolic Syndrome (MS) definition is based on a cluster of metabolic risk factors that identifies subjects at high risk for forthcoming type 2 diabetes mellitus and atherosclerotic cardiovascular diseases (CVD). Although the exact aetiology of the MS still remains unclear, it is known to involve complex interactions between genetic, metabolic and environmental factors, with diet and oxidative stress playing important roles. Regular increased fructose consumption has been associated to some metabolic adverse changes observed in the MS and, thus, fructose-fed is considered a suitable animal model of diet-induced MS. On the other hand, calcium, magnesium and potassium, generally deficient in MS-inducing diets, and abundant in natural mineral-rich waters, have been proposed protective against the MS. Although their exact effects are not yet fully clarified, natural mineral-rich waters present some antioxidant properties that exert protection against reactive oxygen species that are chief contributors to the increase of CVD risk, considering their reactivity to nitric oxide (NO). Such waters also improve some MS metabolic risk factors. Degradation of NO seriously compromises the vasodilatation mechanism leading to endothelial dysfunction, which always precedes atherosclerosis — the main contributor to CVD and erectile dysfunction. Penis erection is a vascular process that strongly depends on NO-induced smooth muscle relaxation. Moreover, NO mediates indirectly the vascular endothelial growth factor (VEGF)-induced angiogenesis, which is fundamental to maintain endothelium integrity in the cavernous tissue. VEGF binds specifically to VEGF tyrosine kinase membrane receptors (VEGFR1 and VEGFR2), and crosstalk in vivo with other angiogenic factors such as angiopoietins that compete for binding to the endothelial-specific Tie2 receptor. Previous work from our group demonstrated that long-term consumption of antioxidant-rich beverages modifies the expression of VEGF, Ang1, Ang2 and their receptors VEGFR1, VEGFR2 and Tie2 in the cavernous tissue of the rat, preventing atherosclerosis progression.

Scientific digital imaging in three dimensions such as when using X-ray computed tomography offers a variety of ways to obtain, filter, and quantify data that can produce vastly different results. These opportunities, performed during image acquisition or during the data processing, can include filtering, cropping, and setting thresholds. Quantifying features in these images can be greatly affected by how the above operations are performed. For example, during binarization, setting the threshold too low or too high can change the number of objects as well as their measured diameter. Here, two facets of three-dimensional quantification are explored. The first will focus on investigating the question of how many voxels are needed within an object to have accurate geometric statistics that are due to the properties of the object and not an artifact of too few voxels. These statistics include but are not limited to percent of total volume, volume of the individual object, Feret shape, and surface area. Using simple cylinders as a starting point, various techniques for smoothing, filtering, and other processing steps can be investigated to aid in determining if they are appropriate for a specific desired statistic for a real dataset. The second area of investigation is the influence of post-processing, particularly segmentation, on measuring the damage statistics in high purity Cu. The most important parts of the pathways of processing are highlighted.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

A scheme of photoabsorption spectroscopy based on scanning tunneling microscopy (STM) has been developed by using a supercontinuum light as the wideband light source of a Fourier transform interferometer for spectroscopic measurements. The performance was demonstrated for a sample of GaAs. The proof-of-concept test showed that the use of the supercontinuum light instead of halogen lamps greatly enhances the signal-to-noise ratio due to the high brilliance of the supercontinuum light emitted from a small core of the photonic crystal fiber that enables tight focusing of the spectroscopy light onto the sample beneath the STM tip.

In this study the nanomorphology of P3HT:PC61BM absorber layers of organic solar cells was studied as a function of the processing parameters and for P3HT with different molecular weight. For this purpose we apply scanning transmission electron microscopy (STEM) at low electron energies in a scanning electron microscope. This method exhibits sensitive material contrast in the high-angle annular dark-field (HAADF) mode, which is well suited to distinguish materials with similar densities and mean atomic numbers. The images taken with low-energy HAADF STEM are compared with conventional transmission electron microscopy and atomic force microscopy images to illustrate the capabilities of the different techniques. For the interpretation of the low-energy HAADF STEM images, a semiempirical equation is used to calculate the image intensities. The experiments show that the nanomorphology of the P3HT:PC61BM blends depends strongly on the molecular weight of the P3HT. Low-molecular-weight P3HT forms rod-like domains during annealing. In contrast, only small globular features are visible in samples containing high-molecular-weight P3HT, which do not change significantly after annealing at 150°C up to 30 min.

Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this “low energy excitation,” photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

Although imperfect spatial localization in single-molecule object tracking experiments has long been recognized to induce apparent motion in an immobile population of molecules, this effect is often ignored or incorrectly analyzed for mobile molecules. In particular, apparent motion due to positional uncertainty is often incorrectly assigned as a distinct diffusive mode. Here we show that, due to both static and dynamic contributions, positional uncertainty does not introduce a new apparent diffusive mode into trajectories, but instead causes a systematic shift of each measured diffusion coefficient. This shift is relatively simple: a factor of σ2/Δt is added to each diffusion coefficient, where σ is the positional uncertainty length scale and Δt is the time interval between observations. Therefore, by calculating the apparent diffusion coefficients as a function of Δt, it is straightforward to separate the true diffusion coefficients from the effective positional uncertainty. As a concrete demonstration, we apply this approach to the diffusion of the protein fibrinogen adsorbed to a hydrophobic surface, a system that exhibits three distinct modes of diffusion.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

A gray cast iron specimen was investigated by color and chemical etching with optical and atomic force microscopy, and the effect of grain orientation on the effectiveness of etching was examined. It was proven that the grain orientation dependence of chemical and color etching is just the opposite, and that the specimen surface after color etching is not uniformly smooth. Explanation for the layer structure of the color etched iron specimen is given.

We describe a method for measuring the lateral focal spot size of a multiphoton laser scanning microscope (LSM) with unprecedented accuracy. A specimen consisting of an aluminum film deposited on a glass coverslip was brought into focus in a LSM and the laser intensity was then increased enough to perform nanoablation of the metal film. This process leaves a permanent trace of the raster path usually taken by the beam during the acquisition of an optical image. A scanning electron microscope (SEM) was then used to determine the nanoablated line width to high accuracy, from which the lateral spot size and hence resolution of the LSM can be determined. To demonstrate our method, we performed analysis of a multiphoton LSM at various infrared wavelengths, and we report measurements of optical lateral spot size with an accuracy of 20 nm, limited only by the resolution of the SEM.

The recent developments of phase-contrast synchrotron imaging techniques have been of great interest for paleontologists, providing three-dimensional (3D) tomographic images of anatomical structures, thereby leading to new paleobiological insights and the discovery of new species. However, until now, it has not been used on features smaller than 5–7 μm voxel size in fossil bones. Because much information is contained within the 3D histological architecture of bone, including an ontogenetic record, crucial for understanding the paleobiology of fossil species, the application of phase-contrast synchrotron tomography to bone at higher resolutions is potentially of great interest. Here we use this technique to provide new 3D insights into the submicron-scale histology of fossil and recent bones, based on the development of new pink-beam configurations, data acquisition strategies, and improved processing tools. Not only do the scans reveal by nondestructive means all of the major features of the histology at a resolution comparable to that of optical microscopy, they provide 3D information that cannot be obtained by any other method.

In conventional X-ray microtomography (μCT), the three-dimensional (3D) distribution of the attenuation coefficient of X-rays is measured and reconstructed in a 3D volume. As spatial resolution increases, the refraction of X-rays becomes a significant phenomenon in the imaging process. Although this so-called phase contrast was initially a cumbersome feature in lab-based μCT, special phase retrieval algorithms were developed to exploit these effects. Clear advantages in terms of visualization and analysis can be seen when phase retrieval algorithms are applied, including an increased signal-to-noise ratio. In this work, this is demonstrated both on simulated and measured data.

Particles of anthropogenic and biological origin, minerals and gaseous elements are the major constituents of the aerosol. Pollen is a biological component of the aerosol, found in suspension with other pollutants. In recent years, research has been conducted on the crossed effects between non-biological and biological pollutants on allergy worsening in susceptible individuals or on the development of symptoms in non-symptomatic ones. Platanus spp pollen is abundantly present in the atmosphere of Porto in early spring and is known to promote allergic respiratory symptoms. The aim of this study was to characterize possible alterations of the main elements present on the Platanus spp. pollen wall under different atmospheric conditions in the coastal city of Porto, Portugal, using EPMA.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Aluminum-lithium alloys are widespread in the aerospace industry. The new 2099 and 2199 alloys provide improved properties, but their microstructure and texture are not well known. This article describes how state-of-the-art field-emission scanning electron microscopy (FE-SEM) can contribute to the characterization of the 2099 aluminum-lithium alloy and metallic alloys in general. Investigations were carried out on bulk and thinned samples. Backscattered electron imaging at 3 kV and scanning transmission electron microscope imaging at 30 kV along with highly efficient microanalysis permitted correlation of experimental and expected structures. Although our results confirm previous studies, this work points out possible substitutions of Mg and Zn with Li, Al, and Cu in the T1 precipitates. Zinc and magnesium are also present in “rice grain”–shaped precipitates at the grain boundaries. The versatility of the FE-SEM is highlighted as it provides information in the macro- and microscales with relevant details. Its ability to probe the distribution of precipitates from nano- to microsizes throughout the matrix makes FE-SEM an essential technique for the characterization of metallic alloys.

Phosphoprotein phosphatase 1 (PPP1) is the most widely expressed and abundant serine/threonine protein phosphatase. PPP1 regulates a variety of cellular functions such as glycogen metabolism, mitosis and meiosis, cell-cycle arrest, apoptosis, dynamics of actin cytoskeleton, protein synthesis among others. The versatility of the PPP1 catalytic subunit (PPP1C) is achieved by associating with different regulatory subunits that target PPP1C to a particular subcellular compartment determining its substrate specificity and activity. PPP1C is expressed in mammals in three isoforms: PPP1CA, PPP1CB and PPP1CC, each encoded by distinct genes. The PPP1C gene undergoes alternative splicing to originate PPP1CC1 and PPP1CC2 variants. PPP1A and PPP1C1 are expressed virtually in all tissues but are particularly enriched in the brain. The subcellular localization of the endogenous PPP1C isoforms is not fully elucidated, but all isoforms are found in the nucleus and cytoplasm, despite some isoform-specific differences in intranuclear distribution. Furthermore, it is known that PPP1 plays a key role in mitosis where PPP1C isoforms are differentially targeted to specific subcellular structures. However, in previous studies, non-neuronal cells were used as a model system to study PPP1 distribution during the cell cycle. In the studies here described, we used the neuronal-like cell line SH-SY5Y since PPP1 is a crucial protein in several neuronal functions.

Single atoms can be considered as the most basic objects for electron microscopy to test the microscope performance and basic concepts for modeling image contrast. In this work high-resolution transmission electron microscopy was applied to image single platinum, molybdenum, and titanium atoms in an aberration-corrected transmission electron microscope. The atoms are deposited on a self-assembled monolayer substrate that induces only negligible contrast. Single-atom contrast simulations were performed on the basis of Weickenmeier-Kohl and Doyle-Turner form factors. Experimental and simulated image intensities are in quantitative agreement on an absolute intensity scale, which is provided by the vacuum image intensity. This demonstrates that direct testing of basic properties such as form factors becomes feasible.

Screw implantation inevitably causes microdamage in surrounding bone. However, little is known about the detailed characteristics of microdamage in peri-screw bone. In this study, we developed a method to construct microdamage cast with methylmethacrylate (MMA) and observed the cast using scanning electron microscopy (SEM). In basic fuchsin stained bone sections observed by bright-field and fluorescence microscopy, diffuse damage, cross-hatched damage, and linear cracks were all presented in peri-screw bone. Using MMA casting/SEM method, we found numerous densely packed microcracks in the areas with diffuse damage. The osteocyte canaliculi and the microcracks consisting of diffuse damage had a similar diameter (or width), usually <0.5 μm, but their morphology was largely different. In the area with cross-hatched damage, the orientation of microcracks was similar to that in diffuse damage, but the number was significantly decreased. Many microcracks were thicker than 1 μm and associated with a rough surface. Large linear cracks (∼10 μm in diameter) occurred in different areas. Plenty of microcracks were present on the surface of some linear cracks. In conclusion, the MMA casting/SEM method can demonstrate the three-dimensional morphology of different types of microdamage, particularly the microcracks in diffuse damage, which are unable to be shown by light microscopy.

In this series of articles, a method is presented that performs (semi)quantitative phase analysis for nanocrystalline transmission electron microscope samples from selected area electron diffraction (SAED) patterns. Volume fractions and degree of fiber texture are determined for the nanocrystalline components. The effect of the amorphous component is minimized by empirical background interpolation. First, the two-dimensional SAED pattern is converted into a one-dimensional distribution similar to X-ray diffraction. Volume fractions of the nanocrystalline components are determined by fitting the spectral components, calculated for the previously identified phases with a priori known structures. These Markers are calculated not only for kinematic conditions, but the Blackwell correction is also applied to take into account dynamic effects for medium thicknesses. Peak shapes and experimental parameters (camera length, etc.) are refined during the fitting iterations. Parameter space is explored with the help of the Downhill-SIMPLEX. The method is implemented in a computer program that runs under the Windows operating system. Part I presented the principles, while part II elaborated current implementation. The present part III demonstrates the usage and efficiency of the computer program by numerous examples. The suggested experimental protocol should be of benefit in experiments aimed at phase analysis using electron diffraction methods.