Image processing algorithms were developed and compared with visual assessment from 12 volunteers to quantify the temporal morphological structure of a single Euglena gracilis organism. Representative images of E. gracilis, showing different morphological characteristics from ovate to cylindrical and elongate, were captured with a bright-field video microscopy system. These images were ranked by the volunteers in order from ovate to elongate. The images were analyzed in the spatial and spatial frequency domain, and the order of the images from each analysis was ranked against the visual assessment. The assessment methods agreeing with the volunteer's preferred sequence were an eccentricity measurement (major axis over the sum of the minor axis at three points), the cross correlation of the image without high pass filtering or edge detection, and cross correlation of the power spectral density.

Scanning confocal electron microscopy (SCEM) is a new imaging technique that is capable of depth sectioning with nanometer-scale depth resolution. However, the depth resolution in the optical axis direction (Z) is worse than might be expected on the basis of the vertical electron probe size calculated with the existence of spherical aberration. To investigate the origin of the degradation, the effects of electron energy loss and chromatic aberration on the depth resolution of annular dark-field SCEM were studied through both experiments and computational simulations. The simulation results obtained by taking these two factors into consideration coincided well with those obtained by experiments, which proved that electron energy loss and chromatic aberration cause blurs at the overfocus sides of the Z-direction intensity profiles rather than degrade the depth resolution much. In addition, a deconvolution method using a simulated point spread function, which combined two Gaussian functions, was adopted to process the XZ-slice images obtained both from experiments and simulations. As a result, the blurs induced by energy loss and chromatic aberration were successfully removed, and there was also about 30% improvement in the depth resolution in deconvoluting the experimental XZ-slice image.

Energy-dispersive X-ray fluorescence was employed to test the hypothesis that beverage consumption or mouthwash utilization will change the chemical properties of dental materials and enamel mineral content. Bovine enamel samples (n = 45) each received two cavity preparations (n = 90), each pair filled with one of three dental materials (R: nanofilled composite resin; GIC: glass-ionomer cement; RMGIC: resin-modified GIC). Furthermore, they were treated with three different solutions (S: saliva; E: erosion/Pepsi Twist®; or EM: erosion+mouthwash/Colgate Plax®). It was found that mineral loss in enamel was greater in GICE samples than in RE > RMGICE > RMGICEM > REM > GICEM. An increased percentage of Zr was found in REM indicating organic matrix degradation. Dental materials tested (R, GIC, and RMGIC) were not able to protect adjacent enamel from acid erosion by the soft drink tested. The use of mouthwash promoted protection of enamel after erosion by the soft drink. To avoid chemical dissolution by mouthwashes, protection by resin composites with surface sealants is recommended.

Digitalis purpurea L. and Digitalis thapsi L. (Iberian endemism) are two well known Portuguese species that contain cardiac glycosides. Last year we found some specimens in the Northeast region of Portugal that appeared to us to be natural hybrids between D. purpurea and D. thapsi. In consequence, morphological, genetic and chemical experimental work was performed, involving a minimum of 30 samples of each plant species collected in this geographic area. Preliminary results of this work have been published.

Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca2+ sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca2+ levels. When global Ca2+ responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

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

Three-dimensional (3D) datasets were recorded of gold nanoparticles placed on both sides of silicon nitride membranes using focal series aberration-corrected scanning transmission electron microscopy (STEM). Deconvolution of the 3D datasets was applied to obtain the highest possible axial resolution. The deconvolution involved two different point spread functions, each calculated iteratively via blind deconvolution. Supporting membranes of different thicknesses were tested to study the effect of beam broadening on the deconvolution. It was found that several iterations of deconvolution was efficient in reducing the imaging noise. With an increasing number of iterations, the axial resolution was increased, and most of the structural information was preserved. Additional iterations improved the axial resolution by maximal a factor of 4 to 6, depending on the particular dataset, and up to 8 nm maximal, but also led to a reduction of the lateral size of the nanoparticles in the image. Thus, the deconvolution procedure optimized for the highest axial resolution is best suited for applications where one is interested in the 3D locations of nanoparticles only.

Since their invention, nanomanipulation systems in scanning electron microscopes (SEMs) have provided researchers with an increasing ability to interact with objects at the nanoscale. However, most nanomanipulators that are capable of generating nanometer displacement operate in an open-loop without suitable feedback mechanisms. In this article, a robust and effective tracking framework for visual servoing applications is presented inside an SEM to achieve more precise tracking manipulation and measurement. A subpixel template matching tracking algorithm based on contour models in the SEM has been developed to improve the tracking accuracy. A feed-forward controller is integrated into the control system to improve the response time. Experimental results demonstrate that a subpixel tracking accuracy is realized. Furthermore, the robustness against clutter can be achieved even in a challenging tracking environment.

A sample preparation method is described for enabling direct correlation of site-specific plan-view and cross-sectional transmission electron microscopy (TEM) analysis of individual nanostructures by employing a dual-beam focused-ion beam (FIB) microscope. This technique is demonstrated using Si nanowires dispersed on a TEM sample support (lacey carbon or Si-nitride). Individual nanowires are first imaged in the plan-view orientation to identify a region of interest; in this case, impurity atoms distributed at crystalline defects that require further investigation in the cross-sectional orientation. Subsequently, the region of interest is capped with a series of ex situ and in situ deposited layers to protect the nanowire and facilitate site-specific lift-out and cross-sectioning using a dual-beam FIB microscope. The lift-out specimen is thinned to electron transparency with site-specific positioning to within ∼200 nm of a target position along the length of the nanowire. Using the described technique, it is possible to produce correlated plan-view and cross-sectional view lattice-resolved TEM images that enable a quasi-3D analysis of crystalline defect structures in a specific nanowire. While the current study is focused on nanowires, the procedure described herein is general for any electron-transparent sample and is broadly applicable for many nanostructures, such as nanowires, nanoparticles, patterned thin films, and devices.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to Nan Yao, nyao@Princeton.edu.

Microfluidic devices can provide unique control over both the chemoattractant gradient and the migration environment of the cells. Our work incorporates laser-machined micro and nanofluidic channels into bulk fused silica and cover slip-sized silica wafers. We have designed “open” chemotaxis devices that produce passive chemoattractant gradients without an external micropipette system. Since the migration area is unobstructed, cells can be easily loaded and strategically placed into the devices with a standard micropipette. The reusable monolithic glass devices have integral ports that can generate multiple gradients in a single experiment. We also used cover slip microfluidics for chemotaxis assays. Passive gradients elicited from these cover slips could be readily adapted for high throughput chemotaxis assays. We have also demonstrated for the first time that cells can be recruited into cover slip ports eliciting passive chemoattractant gradients. This proves, in principle, that intravital cover slip configurations could deliver controlled amounts of drugs, chemicals, or pathogens as well as recruit cells for proteomic or histological analysis in living animals while under microscopic observation. Intravital cover slip fluidics will create a new paradigm for in vivo observation of biological processes.

The histological analyzes performed in gonad cells to verify the occurrence of changes in their anatomy is of crucial importance in order to characterize the dynamics of the gonads during the sexual maturation phase of its cells in specimens of wild marine populations. These histological data, together with other reproductive data, can be used in the monitoring of marine environments in which the Solea solea (Ishiba et al., 2008) lives, namely, the coastal waters of the Iberian Peninsula, Africa and Mediterranean sea (Ishiba et al., 2008). Therefore, the main objective of this study was to evaluate the several cellular types and its quantities and anatomies (Ishiba et al., 2008). The study was performed using a total of 19 specimens of the species Solea solea, collected between the months of January and March of 2011 in two different locations of the Portuguese coast (Aveiro and Leixões, Portugal). The gonad samples were fixated in Formalin-acetic acid-alcohol (FAA) and processed in accordance to the standard histology protocol (Culling et al., 1985). The slices of the gonads were colored with Haematoxylin and Eosin (H&E) (used in the study of the general structure of the gonads, as well as in the identification and localization of the different cellular types of the gonad epithelium). As a result of the study, we found 7 different types of gonad cells: I oogonia (It is not photographed ); II a Chromatin nucleolus oocytes (Fig.1. A); II b perinucleolar oocytes (Fig.1. B); III cortical alveoli oocytes (Fig.1. C); IV vitellogenic oocytes (Fig.1. D); V maturing oocyte (Fig.1. E); VI ovulated oocyte (Fig.1. F), (Hibiya, et al., 1995). The different cellular phases were found in all the gonad samples analyzed in this study and the results corresponded to those expected: a decrease in the size in the cells of the first cellular type, the oogonia and an increase in the size in the cells of all other cellular types found in the study. Histological and histochemical analyses of the Solea solea can be used in anatomic and pathological evaluations of the physiological condition of the animals, as stated previously for the muscles, where histological changes could be observed (such as: degeneration and atrophy of the muscle bundles). In the future, the analytical determination of histological and histochemical changes in the gonads of Solea solea, in conjunction with other analytical processes, such as in situ analyzes, can potentially be used to determine and accurately characterize the physiological condition of animals sampled in any natural environment.

A three-dimensional (3D) study of multiphase nanostructures by chemically selective electron tomography combining tomographic approach and energy-filtered imaging is reported. The implementation of this technique at the nanometer scale requires careful procedures for data acquisition, computing, and analysis. Based on the performances of modern transmission electron microscopy equipment and on developments in data processing, electron tomography in the energy-filtered imaging mode is shown to be a very appropriate analysis tool to provide 3D chemical maps at the nanoscale. Two examples highlight the usefulness of analytical electron tomography to investigate inhomogeneous 3D nanostructures, such as multiphase specimens or core-shell nanoparticles. The capability of discerning in a silica-alumina porous particle the two different components is illustrated. A quantitative analysis in the whole specimen and toward the pore surface is reported. This tool is shown to open new perspectives in catalysis by providing a way to characterize precisely 3D nanostructures from a chemical point of view.

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

For over a decade fluorescence microscopy has demonstrated the capacity to achieve single-molecule localization accuracies of a few nanometers, well below the ∼200 nm lateral and ∼500 nm axial resolution limit of conventional microscopy. Yet, only the recent development of new fluorescence labeling modalities, the increase in sensitivity of imaging hardware, and the creation of novel image analysis tools allow for the emergence of single-molecule-based super-resolution imaging techniques. Novel methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy can typically reach a tenfold increase in resolution compared to standard microscopy methods. Their implementation is relatively easy only requiring minimal changes to a conventional wide-field or total internal reflection fluorescence microscope. The recent translation of these two methods into commercial imaging systems has made them further accessible to researchers in biology. However, these methods are still evolving rapidly toward imaging live samples with high temporal resolution and depth. In this review, we recall the roots of single-molecule localization microscopy, summarize major recent developments, and offer perspective on potential applications.

This article deals with uncertainty in the analysis of strain in silicon nanoscale structures and devices using nanobeam electron diffraction (NBED). Specimen and instrument related errors and instabilities and their effects on NBED analysis are addressed using a nanopatterned ultrathin strained silicon layer directly on oxide as a model system. We demonstrate that zero-loss filtering significantly improves the NBED precision by decreasing the diffuse background in the diffraction patterns. To minimize the systematic deviations the acquired data were verified through a reliability test and then calibrated. Furthermore, the effect of strain relaxation by specimen preparation using a FIB is estimated by comparing profiles, which were acquired by analyzing slices of strained structures in a 220-nm-thick region of the sample (invasive preparation) and the entire strained nanostructures, which are embedded in a thicker region of the same sample (noninvasive preparation). Together with the random deviation, the corresponding systematic shift results in a total deviation of ∼1 × 10−3 for NBED analyses, which is employed to estimate the measurement uncertainty in the thinner sample region. In contrast, the strain in the thick sample region is not affected by the preparation; the systematic shift reduces to a minimum, which improves the total deviation by ∼50%.

Observation of growth, synthesis, dynamics, and electrochemical reactions in the liquid state is an important yet largely unstudied aspect of nanotechnology. The only techniques that can potentially provide the insights necessary to advance our understanding of these mechanisms is simultaneous atomic-scale imaging and quantitative chemical analysis (through spectroscopy) under environmental conditions in the transmission electron microscope. In this study we describe the experimental and technical conditions necessary to obtain electron energy loss (EEL) spectra from a nanoparticle in colloidal suspension using aberration-corrected scanning transmission electron microscopy (STEM) combined with the environmental liquid stage. At a fluid path length below 400 nm, atomic resolution images can be obtained and simultaneous compositional analysis can be achieved. We show that EEL spectroscopy can be used to quantify the total fluid path length around the nanoparticle and demonstrate that characteristic core-loss signals from the suspended nanoparticles can be resolved and analyzed to provide information on the local interfacial chemistry with the surrounding environment. The combined approach using aberration-corrected STEM and EEL spectra with the in situ fluid stage demonstrates a plenary platform for detailed investigations of solution-based catalysis.

The microstructure of Cu80Fe10Ni10 (at. %) granular ribbons was investigated by means of three-dimensional field ion microscopy (3D FIM). This ribbon is composed of magnetic precipitates embedded in a nonmagnetic matrix. The magnetic precipitates have a diameter smaller than 5 nm in the as-spun state and are coherent with the matrix. No accurate characterization of such a microstructure has been performed so far. A tomographic characterization of the microstructure of melt spun and annealed Cu80Fe10Ni10 ribbon was achieved with 3D FIM at the atomic scale. A precise determination of the size distribution, number density, and distance between the precipitates was carried out. The mean diameter for the precipitates is 4 nm in the as-spun state. After 2 h at 350°C, there is an increase of the size of the precipitates, while after 2 h at 400°C the mean diameter of the precipitates decreases. Those data were used as inputs in models that describe the magnetic and magnetoresistive properties of this alloy.

The relation between probe size and spatial resolution of backscattered electron (BSE) images was studied. In addition, the effect of the accelerating voltage, the current intensity and the sample geometry and composition were analyzed. An image synthesis method was developed to generate the images from backscattered electron coefficients obtained from Monte Carlo simulations. Spatial resolutions of simulated images were determined with the SMART-J method, which is based on the Fourier transform of the image. The resolution can be improved by either increasing the signal or decreasing the noise of the backscattered electron image. The analyses demonstrate that using a probe size smaller than the size of the observed object (sample features) does not improve the spatial resolution. For a probe size larger than the feature size, the spatial resolution is proportional to the probe size.