Morphology visualization of irregular shape bacteria by electron holography and tomography

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off‐axis electron holography (EH) and 3D reconstruction electron tomography using high‐angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off‐axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing time and the reconstruction artifacts that may arise during tomogram reconstruction.     

STEM tomography for thick biological specimens

Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.     

Three‐dimensional bright‐field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright‐field scanning transmission electron microscopy (BF‐STEM) and in the present report BF‐STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon‐degrading soil bacterium, Delftia acidovorans Cs1‐4. Dual‐axis BF‐STEM enabled high‐resolution 3‐D tomographic recontructions (6–10 nm) visualization of thick (1250 and 1500 nm) sections. The 3‐D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF‐STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual‐axis BF‐STEM tomography to obtain high‐resolution 3‐D images of novel nanostructures in bacterial biofilms. Future work with dual‐axis BF‐STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures.     

Getting started with a scanning transmission electron microscope coupled to a small computer

A scanning transmission electron microscope (STEM) equipped with a laser-heated gun was coupled to a small computer. Several alterations to the commercially obtained parts of this system are described. On-line operation procedures were developed aiming to reduce the amount of radiation of the specimen area of interest. The resulting system is capable of recording information at the 1.0 to 1.5 nm resolution level by taking advantage of the efficiency of a STEM in recording the information and in controlling the irradiation conditions. These features are important in the study of biological material.     

Reconstruction from in-line electron holograms by digital processing

The digitized in-line electron holograms recorded in an HB-5 STEM instrument were reconstructed by computer processing in place of optical reconstruction. The important parameters of the holograms, namely the spherical aberration and defocus, can be obtained from the electron Ronchigrams. The reconstructed image shows some improvement in resolution as compared with the STEM bright field image. The main limitations appear to be mechanical vibration, drift of the specimen, the relatively small number of pixels in the images, and the uncertainties in determining the spherical aberration coefficient and defocus value.     

Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: Implications for tomography of thick biological sections

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 at% osmium in a carbon matrix, it was found that for 1-μm-thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF STEM tomographic tilt series from a stained 600-nm-thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF STEM but significantly less in the BF STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.     

Annular electron energy-loss spectroscopy in the scanning transmission electron microscope

We study atomic-resolution annular electron energy-loss spectroscopy (AEELS) in scanning transmission electron microscopy (STEM) imaging with experiments and numerical simulations. In this technique the central part of the bright field disk is blocked by a beam stop, forming an annular entry aperture to the spectrometer. The EELS signal thus arises only from electrons scattered inelastically to angles defined by the aperture. It will be shown that this method is more robust than conventional EELS imaging to variations in specimen thickness and can also provide higher spatial resolution. This raises the possibility of lattice resolution imaging of lighter elements or ionization edges previously considered unsuitable for EELS imaging.     

Digital acquisition and processing of electron micrographs using a scanning transmission electron microscope

A digital acquisition system that collects multichannel information from a scanning transmission electron microscope (STEM) and its application are described. The hardware comprises (i) single electron counting detectors, (ii) a digital scan generator, (iii) a digital multi-channel on-line processor, (iv) an interface to a minicomputer, and (v) a display system. Experimental results characterizing these components are presented, and their performance is discussed The software includes assembler coded programs for dynamic file maintenance and fast acquisition of image data, a display driver, and FORTRAN coded application programs. The usefulness of digitized STEM is illustrated by a variety of biological applications.     

Discrimination of heavy and light elements in a specimen by use of scanning transmission electron microscopy

Heavier elements have a larger scattering cross-section for elastically scattered electrons than lighter ones. Furthermore, the maximum number of scattered electrons is at higher scattering angles for heavier atoms. These differences can be used, in principle, to distinguish heavy and light elements from each other in dark field Scanning Transmission Electron Microscopy (STEM). We have achieved such discrimination in practice by collecting the electrons in a STEM experiment at two different angles. The information about the elemental composition that these two images together contain is visualized by forming linear combinations of the images which are specific for light and heavy elementsrrespectively. The results are demonstrated for a specimen consisting of platinum grains on a holey carbon film and for granulocytes stained with osmium tetroxide.     

Problems of interpretation in high resolution electron microscopy

Current assumptions in wave aberration theory and specimen scattering theory are reviewed. More quantitative image simulations would be valuable as well as use of a wider range of imaging techniques, particularly STEM. The severe difficulties of high resolution three-dimensional reconstruction are described and illustrated.     

The formation and interpretation of defect images from crystalline materials in a scanning transmission electron microscope.

The technique of scanning transmission electron microscopy (STEM) has been employed usefully in studies of amorphous materials, and the theory of image formation and interpretation in this case has been well developed. Less attention has been given to the practical and theoretical problems associated with the use of STEM for the examination of crystalline materials. In this case the contrast mechanisms are dominated by Bragg diffraction and so they are quite different from those occurring in amorphous substances. In this paper practical techniques for the observation and interpretation of contrast from defects in crystalline materials are discussed. It is shown that whilst images of defects are obtained readily under all typical STEM operating conditions, the form of the image and the information it contains varies with the angle subtended at the specimen by the detector. If this angle is too large significant image modifications relative to the "conventional" transmission electron microscope case may occur and the resolution of the image may degrade. If this angle is too small, then signal to noise considerations make an interpretation of the image difficult. In this paper we indicate how the detector angle may be chosen correctly, and also present techniques for setting up a STEM instrument for imaging a crystalline material containing lattice defects.     

Composition mapping in InGaN by scanning transmission electron microscopy

We suggest a method for chemical mapping that is based on scanning transmission electron microscopy (STEM) imaging with a high-angle annular dark field (HAADF) detector. The analysis method uses a comparison of intensity normalized with respect to the incident electron beam with intensity calculated employing the frozen lattice approximation. This procedure is validated with an In_0.07Ga_0.93N layer with homogeneous In concentration, where the STEM results were compared with energy filtered imaging, strain state analysis and energy dispersive X-ray analysis. Good agreement was obtained, if the frozen lattice simulations took into account static atomic displacements, caused by the different covalent radii of In and Ga atoms. Using a sample with higher In concentration and series of 32 images taken within 42 min scan time, we did not find any indication for formation of In rich regions due to electron beam irradiation, which is reported in literature to occur for the parallel illumination mode. Image simulation of an In_0.15Ga_0.85N layer that was elastically relaxed with empirical Stillinger-Weber potentials did not reveal significant impact of lattice plane bending on STEM images as well as on the evaluated In concentration profiles for specimen thicknesses of 5, 15 and 50 nm. Image simulation of an abrupt interface between GaN and In_0.15Ga_0.85N for specimen thicknesses up to 200 nm showed that artificial blurring of interfaces is significantly smaller than expected from a simple geometrical model that is based on the beam convergence only. As an application of the method, we give evidence for the existence of In rich regions in an InGaN layer which shows signatures of quantum dot emission in microphotoluminescence spectroscopy experiments.     

Methods for specimen thickness determination in electron microscopy

A new method is described for specimen thickness determination in transmission electron microscopy. This is carried out by marking specimens with gold particles and analysing the images of a tilt series by computer. The method makes it possible to distinguish populations of particles on different planes and calculate the distance between the planes with statistical variation. We have applied it to carbon films as test objects and compared the results with those obtained by transverse sectioning, STEM mass measurement, optical density and frequency change of a quartz crystal oscillator. We have then used the method for thickness measurement of multilayered protein crystals and thin sectioned cells.     

Nanofabrication of cylindrical STEM specimen of InGaAs/GaAs quantum dots for 3D-STEM observation

We demonstrate the multiazimuth observation (360 degrees in principle) of InGaAs/GaAs quantum dots (QDs) by means of a 300 kV scanning transmission electron microscope (STEM), where both cross-sectional and plan-view observations are performed on a single STEM specimen for the first time. A cylindrical specimen with a diameter of 200-300 nm including the QD layer inside along the rotation axis was fabricated by the focused ion beam (FIB) technique, with the application of a newly developed mesa-cutting method to adjust the position and angle of the QD layer precisely. The 360 degrees STEM observation is realized by mounting the cylindrical specimen on a holder equipped with a specimen-rotation mechanism. High potential of 3D-STEM observation is briefly presented by showing high contrast images of QDs, dark field images, and moire fringes with various incident angles.     

High-angle triple-axis specimen holder for three-dimensional diffraction contrast imaging in transmission electron microscopy

Electron tomography requires a wide angular range of specimen-tilt for a reliable three-dimensional (3D) reconstruction. Although specimen holders are commercially available for tomography, they have several limitations, including tilting capability in only one or two axes at most, e.g. tilt-rotate. For amorphous specimens, the image contrast depends on mass and thickness only and the single-tilt holder is adequate for most tomographic image acquisitions. On the other hand, for crystalline materials where image contrast is strongly dependent on diffraction conditions, current commercially available tomography holders are inadequate, because they lack tilt capability in all three orthogonal axes needed to maintain a constant diffraction condition over the whole tilt range. We have developed a high-angle triple-axis (HATA) tomography specimen holder capable of high-angle tilting for the primary horizontal axis with tilting capability in the other (orthogonal) horizontal and vertical axes. This allows the user to trim the specimen tilt to obtain the desired diffraction condition over the whole tilt range of the tomography series. To demonstrate its capabilities, we have used this triple-axis tomography holder with a dual-axis tilt series (the specimen was rotated by 90° ex-situ between series) to obtain tomographic reconstructions of dislocation arrangements in plastically deformed austenitic steel foils.     

Scanning reflection image from a solid specimen in the scanning electron microscope with a condenser-objective lens

To achieve the highest resolution in the scanning electron microscope (SEM) or in the scanning transmission electron microscope (STEM), the sample must be mounted in the high-field region of a condenser-objective lens. A secondary electron (SE) image can then be obtained using a collector before the lens. It is also possible to obtain a scanning reflection image by tilting the specimen so that the second half of the condenser-objective lens field deflects the forward-scattered electrons onto the transmission detector beyond the specimen. Experiments were made with an unmodified commercial SEM fitted with a condenser-objective in the upper stage and with a transmission detector, and it was found that the scanning reflection image from a solid sample can provide additional useful information when used in conjunction with the SE image.     

Time-resolved cryotransmission electron microscopy

We describe a new technique, time-resolved cryotransmission electron microscopy (TRC-TEM), that can be used to study changes in microstructure occurring during dynamic processes such as phase transitions and chemical reactions. The sample is prepared on an electron microscope grid maintained at a fixed temperature in a controlled atmosphere. The dynamic process is induced on the grid by a change in pH, salt, or reactant concentration by rapid mixing with appropriate solutions. Alternatively, induction is by rapid change of specimen temperature, or by controlled evaporation of a volatile component. We call such procedures on-the-grid processing. The dynamic process is permitted to run for a defined time and then the thin-film specimen is thermally fixed by plunging into liquid ethane at its freezing point, producing a cryotransmission electron microscopy specimen. By repeating this procedure with varying delays between induction and sample fixation, we can observe transient microstructures. We demonstrate the use of TRC-TEM to study the intermediate structures that form during the transitions between Lα, I_II, and H_II liquid crystalline phases in phospholipid systems. We also identify several other possible applications of the technique.     

Aperture contrast in thick amorphous specimens using scanning transmission electron microscopy

The contrast observed in thick amorphous specimens using a scanning transmission electron microscope (STEM) can be considerably improved by the use of an optimum collector aperture angle. The size of this angle can be calculated by considering the variation of electron current transmitted through the specimen as a function both of the specimen thickness and of the angle of collection subtended at the specimen. Typically these calculations predict optimum angles to be several times the half-width of the elastic scattering distribution, often 10(-1) rad or more. Observations of biological sections of up to 2 micron in thickness using scanning attachments of commercial transmission microscopes have verifie these results at beam voltages of 50, 100 and 200 kV. Wide angle convergent beam diffraction patterns were used to give accurate values of the effective angles represented by the various collector apertures. Once the linearity of the detector-amplifier system had been established, operation in a line modulation mode enabled quantitative measurements to be made of the image contrast. Such measurements also offer a quick effective method of comparing electron beam penetrations.     

Automated high-throughput electron tomography by pre-calibration of image shifts

Electron tomography is a versatile method for obtaining three‐dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100–500 nm thick) with 4–20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several µm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five‐fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus‐change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.