Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell’s boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a “ground truth” for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.

Biocompatibility testing of new materials is often performed in vitro by measuring the growth rate of mammalian cancer cells in time-lapse images acquired by phase contrast microscopes. The growth rate is measured by tracking cell coverage, which requires an accurate automatic segmentation method. However, cancer cells have irregular shapes that change over time, the mottled background pattern is partially visible through the cells and the images contain artifacts such as halos. We developed a novel algorithm for cell segmentation that copes with the mentioned challenges. It is based on temporal differences of consecutive images and a combination of thresholding, blurring, and morphological operations. We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator, and finally provided comparison with other freely available methods. We propose a new, fully automated method for measuring the cell growth rate based on fitting a coverage curve with the Verhulst population model. The algorithm is fast and shows accuracy comparable with manual segmentation. Most notably it can correctly separate live from dead cells.

Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights the great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.

A reproducible way to transfer a single crystalline sample into a gas environmental cell holder for in situ transmission electron microscopic (TEM) analysis is shown in this study. As in situ holders have only single-tilt capability, it is necessary to prepare the sample precisely along a specific zone axis. This can be achieved by a very accurate focused ion beam lift-out preparation. We show a step-by-step procedure to prepare the sample and transfer it into the gas environmental cell. The sample material is a GaP/Ga(NAsP)/GaP multi-quantum well structure on Si. Scanning TEM observations prove that it is possible to achieve atomic resolution at very high temperatures in a nitrogen environment of 100,000 Pa.

Electron-excited X-ray microanalysis performed with scanning electron microscopy and energy-dispersive spectrometry (EDS) has been used to measure trace elemental constituents of complex multielement materials, where “trace” refers to constituents present at concentrations below 0.01 (mass fraction). High count spectra measured with silicon drift detector EDS were quantified using the standards/matrix correction protocol embedded in the NIST DTSA-II software engine. Robust quantitative analytical results for trace constituents were obtained from concentrations as low as 0.000500 (mass fraction), even in the presence of significant peak interferences from minor (concentration 0.01≤C≤0.1) and major (C>0.1) constituents. Limits of detection as low as 0.000200 were achieved in the absence of peak interference.

Acquiring an atomic-resolution compositional map of crystalline specimens has become routine practice, thus opening possibilities for extracting subatomic information from such maps. A key challenge for achieving subatomic precision is the improvement of signal-to-noise ratio (SNR) of compositional maps. Here, we report a simple and reliable solution for achieving high-SNR energy-dispersive X-ray (EDX) spectroscopy spectrum images for individual atomic columns. The method is based on standard cross-correlation aided by averaging of single-column EDX maps with modifications in the reference image. It produces EDX maps with minimal specimen drift, beam drift, and scan distortions. Step-by-step procedures to determine a self-consistent reference map with a discussion on the reliability, stability, and limitations of the method are presented here.

A microchannel plate was used as an ion sensitive detector in a commercial helium ion microscope (HIM) for dark-field transmission imaging of nanomaterials, i.e. scanning transmission ion microscopy (STIM). In contrast to previous transmission HIM approaches that used secondary electron conversion holders, our new approach detects forward-scattered helium ions on a dedicated annular shaped ion sensitive detector. Minimum collection angles between 125 mrad and 325 mrad were obtained by varying the distance of the sample from the microchannel plate detector during imaging. Monte Carlo simulations were used to predict detector angular ranges at which dark-field images with atomic number contrast could be obtained. We demonstrate atomic number contrast imaging via scanning transmission ion imaging of silica-coated gold nanoparticles and magnetite nanoparticles. Although the resolution of STIM is known to be degraded by beam broadening in the substrate, we imaged magnetite nanoparticles with high contrast on a relatively thick silicon nitride substrate. We expect this new approach to annular dark-field STIM will open avenues for more quantitative ion imaging techniques and advance fundamental understanding of underlying ion scattering mechanisms leading to image formation.

Grain boundaries (GB) are characterized by disorientation of the neighboring grains and the direction of the boundary plane between them. A new approach presented here determines the projection of GB that can be used to determine the latter one. The novelty is that an additional parameter of GB is quantified in addition to the ones provided by the orientation maps, namely the width of the projection of the GB is measured from the same set of diffraction patterns that were recorded for the orientation map, without the need to take any additional images. The diffraction patterns are collected in nanobeam diffraction mode in a transmission electron microscope, pixel-by-pixel, from an area containing two neighboring grains and the boundary between them. In our case, the diffraction patterns were recorded using the beam scanning function of a commercially available system (ASTAR). Our method is based on non-negative matrix factorization applied to the mentioned set of diffraction patterns. The method is encoded in a MATLAB environment, making the results easy to interpret and visualize. The measured GB-projection width is used to determine the orientation of the GB-plane, as given in the study by Kiss et al.

The scanning electron microscope provides a platform for subnanometer resolution characterization of material morphology with excellent topographic and chemical contrast dependent on the used detectors. For imaging applications, the predominantly utilized signals are secondary electrons (SEs) and backscattered electrons (BSEs) that are emitted from the sample surface. Recent advances in detector technology beyond the traditional Everhart–Thornley geometry have enabled the simultaneous acquisition and discrimination of SE and BSE signals. This study demonstrates the imaging capabilities of a recently introduced new detector system that consists of the combination of two in-lens (I-L) detectors and one in-column (I-C) detector. Coupled with biasing the sample stage to reduce electron–specimen interaction volumes, this trinity of detector geometry allows simultaneous acquisition of signals to distinguish chemical contrast from topographical changes of the sample, including the identification of surface contamination. The I-C detector provides 4× improved topography, whereas the I-L detector closest to the sample offers excellent simultaneous chemical contrast imaging while not limiting the minimization of working distance to obtain optimal lateral resolution. Imaging capabilities and contrast mechanisms for all three detectors are discussed quantitatively in direct comparison to each other and the conventional Everhart–Thornley detector.

The damage and ion distribution induced in Si by an inductively coupled plasma Xe focused ion beam was investigated by atom probe tomography. By using predefined patterns it was possible to prepare the atom probe tips with a sub 50 nm end radius in the ion beam microscope. The atom probe reconstruction shows good agreement with simulated implantation profiles and interplanar distances extracted from spatial distribution maps. The elemental profiles of O and C indicate co-implantation during the milling process. The presence of small disc-shaped Xe clusters are also found in the three-dimensional reconstruction. These are attributed to the presence of Xe nanocrystals or bubbles that open during the evaporation process. The expected accumulated dose points to a loss of >95% of the Xe during analysis, which escapes undetected.

There are advantages to performing transmission electron backscattering diffraction (tEBSD) in conjunction with focused ion beam-based specimen preparation for atom probe tomography (APT). Although tEBSD allows users to identify the position and character of grain boundaries, which can then be combined with APT to provide full chemical and orientation characterization of grain boundaries, tEBSD can also provide imaging information that improves the APT specimen preparation process by insuring proper placement of the targeted grain boundary within an APT specimen. In this report we discuss sample tilt angles, ion beam milling energies, and other considerations to optimize Kikuchi diffraction pattern quality for the APT specimen geometry. Coordinated specimen preparation and analysis of a grain boundary in a Ni-based Inconel 600 alloy is used to illustrate the approach revealing a 50° misorientation and trace element segregation to the grain boundary.

There has been increasing interest in second harmonic generation (SHG) imaging approaches for the investigation of atherosclerosis due to the deep penetration and three-dimensional sectioning capabilities of the nonlinear optical microscope. Atherosclerosis involves remodeling or alteration of the collagenous framework in affected vessels. The disease is often characterized by excessive collagen deposition and altered collagen organization. SHG has the capability to accurately characterize collagen structure, which is an essential component in understanding atherosclerotic lesion development and progression. As a structure-based imaging modality, SHG is most impactful in atherosclerosis evaluation in conjunction with other, chemically specific nonlinear optics (NLO) techniques to identify additional components of the lesion. These include the use of coherent anti-Stokes Raman scattering and two-photon excitation fluorescence for studying atherosclerosis burden, and application of stimulated Raman scattering to image cholesterol crystals. However, very few NLO studies have attempted to quantitate differences in control versus atherosclerotic states or to correlate the application to clinical situations. This review highlights the potential of SHG imaging to directly and indirectly describe atherosclerosis as a pathological condition.

The quantity and quality of collagen fibrils in the extracellular matrix (ECM) have a pivotal role in dictating biological processes. Several collagen-binding proteins (CBPs) are known to modulate collagen deposition and fibril diameter. However, limited studies exist on alterations in the fibril ultrastructure by CBPs. In this study, we elucidate how the collagen receptor, discoidin domain receptor 1 (DDR1) regulates the collagen content and ultrastructure in the adventitia of DDR1 knock-out (KO) mice. DDR1 KO mice exhibit increased collagen deposition as observed using Masson’s trichrome. Collagen ultrastructure was evaluated in situ using transmission electron microscopy, scanning electron microscopy, and atomic force microscopy. Although the mean fibril diameter was not significantly different, DDR1 KO mice had a higher percentage of fibrils with larger diameter compared with their wild-type littermates. No significant differences were observed in the length of D-periods. In addition, collagen fibrils from DDR1 KO mice exhibited a small, but statistically significant, increase in the depth of the fibril D-periods. Consistent with these observations, a reduction in the depth of D-periods was observed in collagen fibrils reconstituted with recombinant DDR1-Fc. Our results elucidate how DDR1 modulates collagen fibril ultrastructure in vivo, which may have important consequences in the functional role(s) of the underlying ECM.

Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman’s correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia.

Nucleolar assembly is a cellular event that requires the synthesis and processing of ribosomal RNA, in addition to the participation of pre-nucleolar bodies (PNBs) at the end of mitosis. In mammals and plants, nucleolar biogenesis has been described in detail, but in unicellular eukaryotes it is a poorly understood process. In this study, we used light and electron microscopy cytochemical techniques to investigate the distribution of nucleolar components in the pathway of nucleolus rebuilding during closed cell division in epimastigotes of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis. Silver impregnation specific for nucleolar organizer regions and an ethylenediaminetetraacetic acid regressive procedure to preferentially stain ribonucleoprotein revealed the conservation and dispersion of nucleolar material throughout the nucleoplasm during cell division. Furthermore, at the end of mitosis, the argyrophilic proteins were concentrated in the nucleolar organizer region. Unexpectedly, accumulation of nucleolar material in the form of PNBs was not visualized. We suggest that formation of the nucleolus in epimastigotes of T. cruzi occurs by a process that does not require the concentration of nucleolar material within intermediate nuclear bodies such as mammalian and plant PNBs.

Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool.

The aim of the present work was to investigate birefringence and morphology of the secretory-stage enamel organic extracellular matrix (EOECM), and structural and mechanical properties of mature enamel of upper incisors from adult rats that had been treated with pamidronate disodium (0.5 mg/kg/week for 56 days), using transmitted polarizing and bright-field light microscopies (TPLM and BFLM), energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM) and microhardness testing. BFLM showed no morphological changes of the EOECM in pamidronate and control groups, but TPLM revealed a statistically significant reduction in optical retardation values of birefringence brightness of pamidronate-treated rats when compared with control animals (p<0.01). EDX analysis showed that pamidronate-induced statistically significant decrease in phosphorus’ quantity in outer mature enamel (p<0.01) and an increase in the calcium/phosphorus ratio in that structure (p<0.05). Pamidronate did not induce ultra-structural alterations in mature enamel as revealed by SEM and did not cause a reduction in its microhardness (p>0.05). The present study indicates that pamidronate can affect birefringence of the secretory-stage EOECM, which does not seem to be associated with significant changes in morphological and/or mechanical properties of mature enamel.

An effective method for detecting and characterizing counterfeit finished dosage forms and packaging materials is described in this study. Using attenuated total internal reflection Fourier transform infrared spectroscopic imaging, suspect tablet coating and core formulations as well as multi-layered foil safety seals, bottle labels, and cigarette tear tapes were analyzed and compared directly with those of a stored authentic product. The approach was effective for obtaining molecular information from structures as small as 6 μm.

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e−/Å2 toward 103 e−/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.