Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 10¹⁰ Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography ( Wang et al. (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc . 197, 80–93) or spectroscopic analysis.     

Quantitative STEM mass measurement of biological macromolecules in a 300 kV TEM

For almost four decades, the scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold field‐emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can also provide accurate STEM mass measurements. Using the recently published database of elastic‐scattering cross sections from the National Institute of Standards and Technology, we show that the measured absolute mass values for tobacco mosaic virus and limpet hemocyanin didecamers agree with the known values to within better than 10%. Applying the established approach, whereby tobacco mosaic virus is added to a specimen as a calibration standard, we find that the measured molecular weight of the hemocyanin assemblies agrees with the known value to within 3%. This accuracy is achievable although only a very small fraction (∼0.002) of the incident probe current of 300 kV electrons is scattered onto the annular dark‐field STEM detector. FEG TEMs operating at intermediate voltages (200–400 kV) are becoming common tools for determining the structure of frozen hydrated protein assemblies. The ability to perform mass determination with the same instrument can provide important complementary information about the numbers of subunits comprising the protein assemblies whose structure is being studied.     

Transfer,observation and analysis of frozen hydrated specimens

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit.     

A simple technique for examining frozen hydrated specimens in the scanning electron microscope.

The use of a wide angle backscattered electron detector in a scanning electron microscope, which has the capability of the specimen chamber pressure being controlled independently of the column pressure, provides a simple technique for examining frozen hydrated specimens. Large specimens have been examined within 1 min of being placed on the stub and have been examined for many hours without charging artefacts or distortion due to dehydration.     

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques

Over the last two decades, several different preparative techniques have been developed to investigate frozen‐hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high‐pressure freezing with plunge freezing, and block faces with frozen‐hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high‐pressure freezing proved optimal for high‐resolution studies and provided the best ultrastructural preservation. A combination of these fast‐freezing techniques with cryo‐ultramicrotomy yielded well‐preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo‐sections, and – after evaporating a heavy metal and carbon onto the surface – are stable enough in the electron beam to provide high‐resolution images of large surface areas for statistical analysis in a cryo‐SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen‐hydrated material.     

A new cryo‐STEM method for imaging food colloids in the scanning electron microscope

A new cryo‐scanning transmission electron microscopy (cryo‐STEM) technique for imaging casein micelles in a field emission scanning electron microscope is presented. Thin films of micellar casein suspensions on lacey carbon grids were prepared using a modified sample holder developed by Gatan UK. Bright and dark field images were obtained at ?135°C showing casein micelles in their frozen hydrated state and in the size range 30–500 nm. Results were compared favorably with published images of casein micelles obtained with conventional cryo‐transmission electron microscopy, suggesting that cryo‐STEM is a useful alternative technique for visualizing food colloids close to their native state. SCANNING 32: 150–154, 2010. © 2010 Wiley Periodicals, Inc.     

Non‐rigid alignment in electron tomography in materials science

Electron tomography is a key technique that enables the visualization of an object in three dimensions with a resolution of about a nanometre. High‐quality 3D reconstruction is possible thanks to the latest compressed sensing algorithms and/or better alignment and preprocessing of the 2D projections. Rigid alignment of 2D projections is routine in electron tomography. However, it cannot correct misalignments induced by (i) deformations of the sample due to radiation damage or (ii) drifting of the sample during the acquisition of an image in scanning transmission electron microscope mode. In both cases, those misalignments can give rise to artefacts in the reconstruction. We propose a simple‐to‐implement non‐rigid alignment technique to correct those artefacts. This technique is particularly suited for needle‐shaped samples in materials science. It is initiated by a rigid alignment of the projections and it is then followed by several rigid alignments of different parts of the projections. Piecewise linear deformations are applied to each projection to force them to simultaneously satisfy the rigid alignments of the different parts. The efficiency of this technique is demonstrated on three samples, an intermetallic sample with deformation misalignments due to a high electron dose typical to spectroscopic electron tomography, a porous silicon sample with an extremely thin end particularly sensitive to electron beam and another porous silicon sample that was drifting during image acquisitions.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Thick specimens in the CEM and STEM. Resolution and image formation.

A theory of resolution and image formation is presented for thick amorphous specimens in transmission electron microscopes. Eight modes of operation are considered, four in the scanning transmission electron microscope (STEM) and four in the conventional electron microscope (CEM). A thick specimen is defined here as one in which the resolution of detail is limited by plural scattering of the electron beam. In practice this includes films on the order of a micron in thickness. An analytic theory of plural incoherent scattering is developed which is general with respect to material and beam voltage. The theory gives the distribution of elastically scattered electrons as a function of transverse coordinate and angles, and is directly applicable to optical systems. The theory applies to all thicknesses normally encountered, and includes thin specimens as well as thick specimens. Criteria are proposed for evaluation of the quality of microscope images, and the modulation transfer function is applied to determine some practical estimates of picture quality. The STEM is found to have distinct advantages over the CEM for thick specimens. For a carbon specimen one micron thick a STEM operating in bright field at 90 keV produces an image which is roughly equivalent to that of a CEM operating in bright field at 1 MeV. Improvement can be obtained in the CEM by filtering out eneryg-loss electrons which degrade resolution due to chromatic aberration. This results in a reduction in signal intensity and usable thickness, however.     

The cutting of ultrathin sections with the thickness less than 20 nm from biological specimens embedded in resin blocks

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post‐fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512–517, 2016. © 2016 Wiley Periodicals, Inc.     

Enhancement of contrast in living and fixed specimens by the use of fibre optics

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.     

Recent developments in analytical electron microscopy

Recent years have seen the way in which the analytical electron microscope has been applied to problems involving thin specimens in metallurgy, mineralogy and many branches of biology. The limits of sensitivity have been explored and its potential usefulness in these fields investigated. Problems concerning the interaction of electrons with the specimen are discussed in relation to the correct choice of operating conditions and specimen preparation. In biological work, frozen sections provide new information about subcellular elemental localization of mobile electrolytes, while analysis of tissue prepared by conventional means is used to detect physiological levels of some naturally occurring elements. Examination of mineral dispersions provides analysis of particles just 10 nm thick, visible only in transmission electron microscopy, and further work with thin metal foils confirms the value of high resolution transmission imaging as a complementary facility to micro-analysis. Work has been done to investigate the possibilities of improving sensitivities both by changing operational parameters and instrumental design, and the value of quantitation in thin specimen analysis explored.     

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.     

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.     

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.     

Elemental mapping with elastically scattered electrons

We describe a technique for efficient, quantitative, standardless elemental mapping using a high-angle annular detector in a scanning transmission electron microscope (STEM) to collect elastically scattered electrons. With a single crystal specimen, contrast due to thickness variations, diffraction, and channelling effects can be avoided, so that the resulting image contrast quantitatively reflects variations in impurity concentration. We compare a number of simple analytical approximations to the elastic scattering cross sections and show that a standardless analysis is possible over a wide range of atomic number and inner detector angle to an absolute accuracy of better than 20%.     

A method of normalizing cathodoluminescence images of electron transparent foils for thickness contrast applied to InGaN quantum wells

The uniformity of panchromatic cathodoluminescence (CL) from In_0.09Ga_0.91N/GaN quantum wells at 100 K was investigated using a combined transmission electron microscope–cathodoluminescence instrument. A technique for correcting CL images of electron‐transparent wedge specimens for thickness contrast artefacts is presented. The foil thickness was estimated using the dynamical formulation of the relationship between the thickness and the (experimentally observed) transmitted electron intensity. For a given thickness the CL intensity was calculated using the Everhart–Hoff depth‐dose function and also taking into account surface recombination losses. Experimental CL images were normalized by dividing by the calculated CL value at each point. The procedure was successful in calculating the underlying materials contrast in CL images of thin specimens of InGaN single quantum wells. Non‐uniformities in the CL emission on the scale of ～0.7 µm were observed.     

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.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.