Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging

As techniques for electron energy‐loss spectroscopy (EELS) reach a higher degree of optimization, experimental detection limits for analysing biological structures are approaching values predicted by the physics of the electron scattering. Theory indicates that it should be possible to detect a single atom of certain elements like calcium and iron contained in a macromolecular assembly using a finely focused probe in the scanning transmission electron microscope (STEM). To test this prediction, EELS elemental maps have been recorded with the spectrum‐imaging technique in a VG Microscopes HB501 STEM coupled to a Gatan Enfina spectrometer, which is equipped with an efficient charge‐coupled device (CCD) array detector. By recording spectrum‐images of haemoglobin adsorbed onto a thin carbon film, it is shown that the four heme groups in a single molecule can be detected with a signal‐to‐noise ratio of ～10 : 1. Other measurements demonstrate that calcium adsorbed onto a thin carbon film can be imaged at single atom sensitivity with a signal‐to‐noise ratio of ～5 : 1. Despite radiation damage due to the necessarily high electron dose, it is anticipated that mapping single atoms of metals and other bound elements will find useful applications in characterizing large protein assemblies.     

Analysis of directly frozen macromolecules and tissues in the field-emission STEM

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (? 10³ e/nm²) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm². For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm² regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.     

Free-standing graphene by scanning transmission electron microscopy

Free-standing graphene sheets have been imaged by scanning transmission electron microscopy (STEM). We show that the discrete numbers of graphene layers enable an accurate calibration of STEM intensity to be performed over an extended thickness and with single atomic layer sensitivity. We have applied this calibration to carbon nanoparticles with complex structures. This leads to the direct and accurate measurement of the electron mean free path. Here, we demonstrate potentials using graphene sheets as a novel mass standard in STEM-based mass spectrometry.     

Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography

By combining electron tomography with energy-filtered electron microscopy, we have shown the feasibility of determining the three-dimensional distributions of phosphorus in biological specimens. Thin sections of the nematode, Caenorhabditis elegans were prepared by high-pressure freezing, freeze-substitution and plastic embedding. Images were recorded at energy losses above and below the phosphorus L2,3 edge using a post-column imaging filter operating at a beam energy of 120 keV. The unstained specimens exhibited minimal contrast in bright-field images. After it was determined that the specimen was sufficiently thin to allow two-window ratio imaging of phosphorus, pairs of pre-edge and post-edge images were acquired in series over a tilt range of +/-55 degrees at 5 degrees increments for two orthogonal tilt axes. The projected phosphorus distributions were aligned using the pre-edge images that contained inelastic contrast from colloidal gold particles deposited on the specimen surface. A reconstruction and surface rendering of the phosphorus distribution clearly revealed features 15-20 nm in diameter, which were identified as ribosomes distributed along the stacked membranes of endoplasmic reticulum and in the cytoplasm. The sensitivity of the technique was estimated at < 35 phosphorus atoms per voxel based on the known total ribosomal phosphorus content of approximately 7000 atoms. Although a high electron dose of approximately 10(7)e/nm2 was required to record two-axis tilt series, specimens were sufficiently stable to allow image alignment and tomographic reconstruction.     

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.     

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10³ e/nm²) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10¹⁰ e/nm²) is required resulting in mass loss that may be attributable to ‘knock-on’ radiation damage.     

Gentle STEM: ADF imaging and EELS at low primary energies

Aberration correction of the scanning transmission electron microscope (STEM) has made it possible to reach probe sizes close to 1 Å at 60 keV, an operating energy that avoids direct knock-on damage in materials consisting of light atoms such as B, C, N and O. Although greatly reduced, some radiation damage is still present at this energy, and this limits the maximum usable electron dose. Elemental analysis by electron energy loss spectroscopy (EELS) is then usefully supplemented by annular dark field (ADF) imaging, for which the signal is larger. Because of its strong Z dependence, ADF allows the chemical identification of individual atoms, both heavy and light, and it can also record the atomic motion of individual heavy atoms in considerable detail. We illustrate these points by ADF images and EELS of nanotubes containing nanopods filled with single atoms of Er, and by ADF images of graphene with impurity atoms.     

Applications of medium-voltage STEM for the 3-D study of organelles within very thick sections

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3–8 μm) of Epon-embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-μm-thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e^—/Å₂/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4-μm-thick sections with a spot-size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.     

Phosphorus detection in vitrified bacteria by cryo‐STEM annular dark‐field analysis

Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low‐dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus‐containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.     

Visibility and stability of a 12-tungsten atom complex in the scanning transmission electron microscope

A complex consisting of 12 tungsten atoms has been studied in terms of signal-to-noise (S/N) and dose response in the scanning transmission electron microscope (STEM), to evaluate its suitability for use as a approximately 1 nm resolution biological label. Molecular weight of the complex was measured as a function of radius of integration, and results were in agreement with the calculated formula weight. S/N was highest at the lowest radius of integration (0.25 nm), and decreased monotonically with increasing radius. The complex was clearly visible at a dose of 4 X 10(3) e/nm2, and exhibited negligible mass loss (approximately 8%) after an accumulated dose of 1.28 X 10(5) e/nm2. Beam-induced motion was small, 0.46 nm rms after 4 X 10(4) e/nm2. Some intensity fluctuations were observed between successive scans of the same clusters, for which a diffraction-based explanation is advanced. Upon suitable functionalization, the tungsten complex is expected to complement the undecagold cluster already in use for site-specific labeling.     

Prospects for 3D, nanometer-resolution imaging by confocal STEM

Confocal STEM is a new electron microscopy imaging mode. In a microscope with spherical aberration-corrected electron optics, it can produce three-dimensional (3D) images by optical sectioning. We have adapted the linear imaging theory of light confocal microscopy to confocal STEM and use it to suggest optimum imaging conditions for a confocal STEM limited by fifth-order spherical aberration. We predict that current or near-future microscopes will be able to produce 3D images with 1 nm vertical resolution and sub-Angstrom lateral resolution. Multislice simulations show that we will need to be cautious in interpreting these images, however, as they can be complicated by dynamical electron scattering.     

Imaging of radiation-sensitive samples in transmission electron microscopes equipped with Zernike phase plates

We have optimized a bright-field transmission electron microscope for imaging of high-resolution radiation-sensitive materials by calculating the imaging dose n(0) needed to obtain a signal-to-noise ratio (SNR)=5. Installing a Zernike phase plate (ZP) decreases the dose needed to detect single atoms by as much as a factor of two at 300 kV. For imaging larger objects, such as Gaussian objects with full-width at half-maximum larger than 0.15 nm, ZP appears more efficient in reducing the imaging dose than correcting for spherical aberration. The imaging dose n(0) does not decrease with extending of chromatic resolution limit by reducing chromatic aberration, using high accelerating potential (U(0)=300 kV), because the image contrast increases slower than the reciprocal of detection radius. However, reducing chromatic aberration would allow accelerating potential to be reduced leading to imaging doses below 10 e(-)/A(2) for a single iodine atom when a CS-corrector and a ZP are used together. Our simulations indicate that, in addition to microscope hardware, optimization is heavily dependent on the nature of the specimen under investigation.     

First experimental test of a new monochromated and aberration-corrected 200 kV field-emission scanning transmission electron microscope

The first 200 kV scanning transmission electron microscope (STEM) with an imaging energy filter, a monochromator and a corrector for the spherical aberration (Cs-corrector) of the illumination system has been built and tested. The STEM/TEM concept with Koehler illumination allows to switch easily between STEM mode for analytical and TEM mode for high-resolution or in situ studies. The Cs-corrector allows the use of large illumination angles for retaining a sufficiently high beam current despite the intensity loss in the monochromator. With the monochromator on and a 3 microm slit in the dispersion plane that gives 0.26 eV full-width at half-maximum (FWHM) energy resolution we have obtained so far an electron beam smaller than 0.20 nm in diameter (FWHM as measured by scanning the spot quickly over the CCD) which contains 7 pA current and, according to simulations, should be around 0.12 nm in true size. A high-angle annular dark field (ADF) image with isotropic resolution better than 0.28 nm has been recorded with the monochromator in the above configuration and the Cs-corrector on. The beam current is still somewhat low for electron energy-loss spectroscopy (EELS) but is expected to increase substantially by optimising the condenser set-up and using a somewhat larger condenser aperture.     

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.     

Catalyst particle sizes from Rutherford scattered intensities

We show that the number of atoms in a small supported catalyst cluster can be estimated from the strength of electron scattering into a high angle annular detector in the STEM. The technique is related to the Z contrast methods developed by Crewe, Wall, Langmore and Isaacson. It works best for high atomic number catalyst particles when supported on low atomic number supports, such as Pt on γ-aluminium oxide. The method is particularly useful for detecting and measuring particles in the sub-nanometre size range where bright field images are unreliable. Unlike the Z contrast methods, a high angle annular detector is used, which avoids intensity modulations arising from Bragg reflections. The signal is mostly high angle diffuse scattering, which is predominantly Rutherford scattering, and is proportional to the number of atoms probed by the beam, weighted by their individual scattering cross-sections. Scattering strengths of individual clusters are computed from digitized high angle annular detector images. Data for Pt on γ-aluminium oxide, when plotted as imaged area^1/2 against intensity^1/3, define a straight line. Such plots provide calibration of the intensity increment per atom without the need of external calibration, although assumptions about particle morphology must be made. Reliable results require high signal-to-noise and optimum sampling of the specimen. For an STEM probe size of 0.35 nm, Pt clusters containing as few as three atoms can be detected when supported on typical, 20 nm thick, γ-aluminium oxide supports.     

Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells.

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.     

Fluctuation microscopy in the STEM

Fluctuation electron microscopy is a technique for studying medium-range order in disordered materials. We present an implementation of fluctuation microscopy using nanodiffraction in a scanning transmission electron microscope (STEM) at a spatial resolution varying from 0.8 to 5.0 nm. Compared to conventional TEM (CTEM), the STEM-based technique offers a denser scattering vector sampling at a reduced sample dose and easier access to variable resolution information. We have reproduced results on amorphous silicon previously obtained by CTEM-based fluctuation microscopy, and report initial variable-resolution measurements on amorphous germanium.     

Johns Hopkins University, Baltimore, Maryland

The STEM can be used in one of three modes: 1) to image individual atoms; 2) to measure mass or molecular weight; 3) to collect electron energy loss spectra or x-ray fluorescence data. Heavy atom imaging is used to identify chemical groups in a molecule or macromolecules in an assembly. Specific labels have been developed for bases in nucleic acids. These permit localization of bound proteins on single strand nucleic acids. Pt(gly-L-met)Cl is a specific label for methionine residues of proteins as shown with the SLS aggregate of collagen. Lysine can be labeled as well if first methyl (methyl-thio-acetimidate) is coupled. This labeling procedure permits the localization of individual histones within a nucleosome. Mass determination can be used to answer crucial questions about biological assemblies. This is demonstrated by examples from muscle structure.     

Three-dimensional imaging in double aberration-corrected scanning confocal electron microscopy, part II: inelastic scattering

The implementation of spherical aberration-corrected pre- and post-specimen lenses in the same instrument has facilitated the creation of sub-Angstrom electron probes and has made aberration-corrected scanning confocal electron microscopy (SCEM) possible. Further to the discussion of elastic SCEM imaging in our previous paper, we show that by performing a 3D raster scan through a crystalline sample using inelastic SCEM imaging it will be possible to determine the location of isolated impurity atoms embedded within a bulk matrix. In particular, the use of electron energy loss spectroscopy based on inner-shell ionization to uniquely identify these atoms is explored. Comparisons with scanning transmission electron microscopy (STEM) are made showing that SCEM will improve both the lateral and depth resolution relative to STEM. In particular, the expected poor resolution of STEM depth sectioning for extended objects is overcome in the SCEM geometry.     

Interactions of electron beams with surfaces of MgO crystals

The strong interaction of electrons with the flat surfaces of small crystals has been investigated by high resolution CTEM and STEM instruments. When cubic crystals of MgO smoke with edges 20–300 nm are oriented so that the ?001? or ?011? zone axis is parallel to the optical axis, then two kinds of external fringes are observed at (100) surfaces. One kind is parallel to the surface, having spacings up to 0.4 nm. These are caused by interference among the electron channelled along the surface. Fresnel-diffracted ones and the remnant of the incident beam. Fringes of the other kind, which appear as fine structure in the first kind of fringes, are perpendicular to the crystal edge. When an electron beam is parallel to the ?011? axis, the second kind of fringe, whose spacing is 0.3 nm corresponding to d₀₁₁, shows the difference of the surface potential between magnesium atoms and oxygen atoms. Selected area diffraction patterns and microdiffraction patterns also show the same periodicities as in the two kinds of fringes. Simulated images, using the scattering amplitudes for ions, are compared with observations.