Combining cryo-TEM and energy-filtered TEM for imaging organic core-shell nanoparticles and defining the polymer distribution.

Nanoparticulate systems intended for the use in drug delivery are getting more and more complex. Composite nanoparticles, such as core-shell particles are designed in order to be used for co-delivery of drugs or a modified release profile. Often the structure can only be postulated by the preparation process, such as surface polymerization, but cannot be experimentally determined due to a lack of appropriate analytical methods. Here a core-shell particle system composed of two biodegradable and biocompatible materials, gelatin and PLGA, is developed. In order to reveal the actual polymer distribution, a combination of cryo-transmission electron microscopy and energy-filtered transmission electron microscopy was established. Using the occurrence of specific elements in combination with degradation kinetics induced by the electron beam allows to conclude on the nanoparticles' architecture. Based on these methods and thus, the particle composition, the drug delivery system can be further developed.

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps.

We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

LED for hyperspectral imaging - a new selection method.

Hyperspectral imaging (HSI) has become a sophisticated technique in modern applications such as food analyses, recycling technology, medicine, pharmacy and forensic science. It allows one to analyse both spatial and spectral information from an object. But hyperspectral cameras are still expensive due to their extended wavelength range. The development of new light-emitting diodes (LED) in the recent past enables another approach to HSI using a monochrome camera in combination with a LED-based illumination. However, such a system has a lower spectral resolution. Additionally, the growing supply of LED on the market complicates the selection of LED. In this paper, we propose a new time efficient selection method for the design process of an illumination. It chooses an optimised LED combination from an existing database to match a predefined spectral power distribution. Therefore, an algorithm is used to evaluate various LED combinations. Furthermore, the method considers the spectral behaviour of each LED in dependence of forward current and temperature of the solder point. Our method has already shown promise during the selection process for even spectral distributions which is demonstrated in the study. Additionally, we will show its potential for HSI illuminations.

Obtaining 3D Chemical Maps by Energy Filtered Transmission Electron Microscopy Tomography.

Energy filtered transmission electron microscopy tomography (EFTEM tomography) can provide three-dimensional (3D) chemical maps of materials at a nanometric scale. EFTEM tomography can separate chemical elements that are very difficult to distinguish using other imaging techniques. The experimental protocol described here shows how to create 3D chemical maps to understand the chemical distribution and morphology of a material. Sample preparation steps for data segmentation are presented. This protocol permits the 3D distribution analysis of chemical elements in a nanometric sample. However, it should be noted that currently, the 3D chemical maps can only be generated for samples that are not beam sensitive, since the recording of filtered images requires long exposure times to an intense electron beam. The protocol was applied to quantify the chemical distribution of the components of two different heterogeneous catalyst supports. In the first study, the chemical distribution of aluminum and titanium in titania-alumina supports was analyzed. The samples were prepared using the swing-pH method. In the second, the chemical distribution of aluminum and silicon in silica-alumina supports that were prepared using the sol-powder and mechanical mixture methods was examined.

Application of EELS and EFTEM to the life sciences enabled by the contributions of Ondrej Krivanek.

The pioneering contributions of Ondrej Krivanek to the development of electron energy loss spectrometers, energy filters, and detectors for transmission and scanning transmission electron microscopes have provided researchers with indispensible tools across a wide range of disciplines in the physical sciences, ranging from condensed matter physics, to chemistry, mineralogy, materials science, and nanotechnology. In addition, the same instrumentation has extended its reach into the life sciences, and it is this aspect of Ondrej Krivanek's influential contributions that will be surveyed here, together with some personal recollections. Traditionally, electron microscopy has given a purely morphological view of the biological structures that compose cells and tissues. However, the availability of high-performance electron energy loss spectrometers and energy filters offers complementary information about the elemental and chemical composition at the subcellular scale. Such information has proven to be valuable for applications in cell and structural biology, microbiology, histology, pathology, and more generally in the biomedical sciences.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI).

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 µm, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Mapping dynamical mechanical properties of osteonal bone by scanning acoustic microscopy in time-of-flight mode.

An important determinant of mechanical properties of bone is Young's modulus and its variation in individual osteons of cortical bone tissue. Its mechanical behavior also depends on deformation rate owing to its visco- or poroelastic properties. We developed a method to measure dynamical mechanical properties of bulk bone tissue at osteonal level based on scanning acoustic microscopy (SAM) using time-of-flight (TOF) measurements in combination with quantitative backscattered electron imaging (qBEI). SAM-TOF yields local sound velocities and qBEI corresponding material densities together providing elastic properties. Osteons (n=55) were measured in three human femoral diaphyseal ground bone sections (∼ 30 µm in thickness). In addition, subchondral bone and mineralized articular cartilage were investigated. The mean mineral contents, the mean sound velocities, and the mean elastic modulus of the osteons ranged from 20 to 26 wt%, from 3,819 to 5,260 m/s, and from 21 to 44 GPa, respectively. There was a strong positive correlation between material density and sound velocity (Pearson's r=0.701; p<0.0001) of the osteons. Sound velocities between cartilage and bone was similar, though material density was higher in cartilage (+4.46%, p<0.0001). These results demonstrate the power of SAM-TOF to estimate dynamic mechanical properties of the bone materials at the osteonal level.

Improving signal to noise in labeled biological specimens using energy-filtered TEM of sections with a drift correction strategy and a direct detection device.

Energy filtered transmission electron microscopy techniques are regularly used to build elemental maps of spatially distributed nanoparticles in materials and biological specimens. When working with thick biological sections, electron energy loss spectroscopy techniques involving core-loss electrons often require exposures exceeding several minutes to provide sufficient signal to noise. Image quality with these long exposures is often compromised by specimen drift, which results in blurring and reduced resolution. To mitigate drift artifacts, a series of short exposure images can be acquired, aligned, and merged to form a single image. For samples where the target elements have extremely low signal yields, the use of charge coupled device (CCD)-based detectors for this purpose can be problematic. At short acquisition times, the images produced by CCDs can be noisy and may contain fixed pattern artifacts that impact subsequent correlative alignment. Here we report on the use of direct electron detection devices (DDD's) to increase the signal to noise as compared with CCD's. A 3× improvement in signal is reported with a DDD versus a comparably formatted CCD, with equivalent dose on each detector. With the fast rolling-readout design of the DDD, the duty cycle provides a major benefit, as there is no dead time between successive frames.

3D multi-slab diffusion-weighted readout-segmented EPI with real-time cardiac-reordered K-space acquisition.

PURPOSE: The aim of this study was to develop, implement, and demonstrate a three-dimensional (3D) extension of the readout-segmented echo-planar imaging (rs-EPI) sequence for diffusion imaging. THEORY AND METHODS: Potential k-space acquisition schemes were assessed by simulating their associated spatial point spread functions. Motion-induced phase artifacts were also simulated to test navigator corrections and a real-time reordering of the k-space acquisition relative to the cardiac cycle. The cardiac reordering strategy preferentially chooses readout segments closer to the center of 3D k-space during diastole. Motion-induced phase artifacts were quantified by calculating the voxel-wise temporal variation in a set of repeated diffusion-weighted acquisitions. Based on the results of these simulations, a 2D navigated multi-slab rs-EPI sequence with real-time cardiac reordering was implemented. The multi-slab implementation enables signal-to-noise ratio-optimal repetition times of 1-2 s. RESULTS: Cardiac reordering was validated in simulations and in vivo using the multi-slab rs-EPI sequence. In comparisons with standard k-space acquisitions, cardiac reordering was shown to reduce the variability due to motion-induced phase artifacts by 30-50%. High-resolution diffusion tensor imaging data acquired with the cardiac-reordered multi-slab rs-EPI sequence are presented. CONCLUSION: A 3D multi-slab rs-EPI sequence with cardiac reordering has been demonstrated in vivo and is shown to provide high-quality 3D diffusion-weighted data sets.

Biological applications of energy-filtered TEM.

The techniques of electron energy-loss spectroscopy (EELS) and energy-filtered TEM (EFTEM) are routinely applied in the physical sciences to map the distribution of elements at the nanoscale. EELS can also provide details of the bonding/valence of elements through variations in the fine structure of elemental peaks in the spectrum. While applications of these techniques in biology are less prevalent, their ability to detect both the light elements (e.g., C, N, O, P, S) that form the building blocks of biological systems and heavier elements (e.g., metals) makes them potentially important techniques for investigating local chemical variations in tissues and cells. Successful application of EELS and EFTEM in biology requires both an understanding of the techniques themselves and expertise in specimen preparation. Care must be taken to avoid the diffusion of elements during the preparation process to avoid artifacts in the resulting element maps. The power of the techniques is demonstrated here using tissue from a marine mollusc (chiton).

Electron spectroscopic tomography of specific chromatin domains.

The eukaryotic genome is packaged within the nucleus as poly-nucleosome 10 nm chromatin fibres. The nucleosome core particle, the fundamental chromatin subunit, consists of a DNA molecule wrapped around a histone octamer. Biochemical modifications of both the DNA and histone proteins have been characterized that influence chromatin structure and function. These modifications include DNA methylation, histone variants and posttranslational modifications of the core histone protein tails. An outstanding area for investigation in the field of nuclear cell biology is the characterization of the functional relation between these biochemical modifications and the underlying chromatin structure and nuclear sub-compartmentalization. Electron spectroscopic tomography is a high-resolution microscopy technique that facilitates visualization of individual 10 nm chromatin fibres in three dimensions. The method, therefore, has a role to play in exploring the relationships of the epigenome and nuclear organization. Correlating immunofluorescence microscopy with electron spectroscopic tomography provides a powerful approach to relate epigenetic marks with high resolution chromatin organization.

The influence of inelastic scattering on EFTEM images--exemplified at 20 kV for graphene and silicon.

We present model-based image simulations for zero-loss and plasmon-loss filtered images at 20 kV for graphene and silicon based on the mutual coherence approach. In addition, a new approximation for the mixed dynamic form factor is introduced. In our calculation multiple elastic scattering and one inelastic scattering are taken into account. The simulation shows that even the intensity of zero-loss filtered image is attenuated by the interference between inelastically scattered waves. Moreover, the intensity of plasmon-loss filtered images cannot be neglected, either.

Contrast in atomically resolved EF-SCEM imaging.

Energy-filtered scanning confocal electron microscopy (EF-SCEM) is a technique that uses the reduced depth of field of an aberration-corrected transmission electron microscope to provide three-dimensional (3D) compositional information. Using a silicon sample in the <110> orientation, we show that EF-SCEM image data can be recorded that shows lattice resolution in the plane perpendicular to the incident beam direction. The confocal effect is demonstrated through the reduction of the mean intensity as the confocal plane is displaced from the sample mid-plane, unlike optical sectioning in high-angle annular dark-field scanning transmission electron microscopy (STEM). Simulations of the EF-SCEM data show agreement with the experimental data, and allow the interpretability of the data to be explored. The effects of channelling, absorption and delocalisation complicate the quantitative and qualitative interpretation of the data, highlighting the need for matching to simulations. Finally the effects of the finite detector pin-hole aperture size are explored, and we show that the EF-SCEM contrast in the plane perpendicular to the beam direction starts to resemble that of a STEM spectrum imaging experiment as the aperture size increases.

Microstructure, chemistry, and electronic structure of natural hybrid composites in abalone shell.

The crystal structure and chemical composition at the inorganic/inorganic and inorganic/organic interfaces in abalone shell (genus Haliotis) were investigated using advanced analytical transmission electron microscopy (TEM) methods. Electron energy-loss near-edge structures (ELNES) of Ca-M2,3, C-K, Ca-L2,3, O-K and low-loss EEL spectra acquired from aragonite and calcite are distinctly different. When comparing biogenic with inorganic material for aragonite, only minor differences in C-K fine structures could be detected. The crystal structure of the mineral bridges was confirmed by ELNES experiments. ELNES and energy-filtered TEM (EFTEM) experiments of regular and self-healed interfaces between nacreous aragonite and prismatic calcite reveal relatively rough transitions. In this work, the importance of TEM specimen preparation and specimen damage on structural features is discussed.

Correlative fluorescence and EFTEM imaging of the organized components of the mammalian nucleus.

The cell nucleus contains many distinct subnuclear compartments, domains, and bodies that vary in their composition, structure, and function. While the cellular constituents that occupy the subnuclear regions may be well known, defining the structural details of the molecular assembly of the constituents has been more difficult. A correlative fluorescence and energy-filtering transmission electron microscopy (EFTEM) imaging method has the ability to provide these details. The correlative microscopy method described here allows the tracking of subnuclear structures from specific cells by fluorescence microscopy and then, using electron energy loss imaging in the transmission electron microscope, reveals the ultrastructural features of the nuclear components along with endogenous elemental information that relates directly to the biochemical composition of the structure. The ultrastructural features and composition of well-characterized PML bodies and interchromatin granule clusters are compared to those of ligand-activated glucocorticoid receptor (GR) foci, with GR foci containing fibrogranular nucleic acid-containing features and PML bodies being devoid of nucleic acid.

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.

Nanoscale mapping of plasmon resonances of functional multibranched gold nanoparticles.

The optical response of multibranched gold nanoparticles is studied by means of electron energy-loss spectroscopy (EELS) in aberration corrected STEM mode. In every case the plasmon response is constant and variations in the maxima positions were found to be dependent on the branches aspect ratio. The good spatial resolution combined with the high energy resolution (0.18 eV) of the monochromated electron beam allows mapping the different plasmonic modes along the entire nanoparticles ranging from 0.7 eV up to 2.25 eV.

Improving the reliability of the background extrapolation in transmission electron microscopy elemental maps by using three pre-edge windows.

Over the last decades, elemental maps have become a powerful tool for the analysis of the spatial distribution of the elements within specimen. In energy-filtered transmission electron microscopy (EFTEM) one commonly uses two pre-edge and one post-edge image for the calculation of elemental maps. However, this so called three-window method can introduce serious errors into the extrapolated background for the post-edge window. Since this method uses only two pre-edge windows as data points to calculate a background model that depends on two fit parameters, the quality of the extrapolation can be estimated only statistically assuming that the background model is correct. In this paper, we will discuss a possibility to improve the accuracy and reliability of the background extrapolation by using a third pre-edge window. Since with three data points the extrapolation becomes over-determined, this change permits us to estimate not only the statistical uncertainly of the fit, but also the systematic error by using the experimental data. Furthermore we will discuss in this paper the acquisition parameters that should be used for the energy windows to reach an optimal signal-to-noise ratio (SNR) in the elemental maps.

Insight of EDX analysis and EFTEM: are spherocrystals located in Strombidae digestive gland implied in detoxification of trace metals?

Digestive tubules of Strombidae are composed by three cell types: digestive cells, vacuolated cells, and crypt cells. The last one is characterized by the presence of intracellular granules identified as spherocrystals. Such structures are known to occur in basophilic cells of gastropod digestive gland, where they are supposed to be involved in the regulation of some minerals and in detoxification. In this study, energy-dispersive X-ray analysis (EDX) and energy filtered transmission electron microscopy (EFTEM) were used to determine the elemental content of spherocrystals in two Strombidae, Strombus gigas and Strombus pugilis. In freshly collected individuals of both species, the following elements were detected: Ca, Fe, Mg, P, and Zn. Aluminum and Mn were also detected in S. gigas. Their presence in spherocrystals indicates that, in Strombidae, spherocrystals are involved in the regulation of minerals and essential trace metals. In order to answer the question "are spherocrystals involved in nonessential trace metals scavenging?," artificial cadmium and lead exposure by both waterborne and dietary pathways was applied to S. pugilis. No evidence of cadmium (Cd(NO(3))(2)) or lead (Pb(NO(3))(2)) provided by food was found in spherocrystals. Cadmium provided in water (Cd(NO(3))(2) and CdCl(2)) causes structural modifications of the digestive gland; however, this element was not trapped in spherocrystals. These results suggest that spherocrystals are not involved in detoxification of such nonessential trace metals.