High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

A longstanding limitation of imaging with serial block‐face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block‐face due to image jitter. Typically, variable‐pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal‐to‐noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block‐face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block‐face ultramicrotome. This system enables the application of nitrogen gas precisely over the block‐face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high‐resolution block‐face imaging of even the most charge prone of epoxy‐embedded biological samples.     

Scanning electron microscopy of nonconductive specimens at critical energies in a cathode lens system

A method for scanning electron microscopy imaging of nonconductive specimens, based on measurement and utilisation of a critical energy, is described in detail together with examples of its application. The critical energy, at which the total electron yield curve crosses the unit level, is estimated on the basis of measurement of the image signal development from the beginning of irradiation. This approach, concentrated onto the detected signal as the only quantity crucial for the given purpose of acquiring a noncharged micrograph, evades consequences of any changes in an irradiated specimen that influence the total electron yield curve and possibly also the critical energy value. Implementation of the automated method, realised using a cathode lens-equipped scanning electron microsope (SEM), enables one to establish a mean rate of charging over the field of view and its dependence on the electron landing energy. This dependence enables one to determine the energy of a minimum damage of the image of the given field of view. Factors influencing reliability and applicability of the method are discussed and examples of noncharged micrographs of specimens from both life and material science fields are presented.     

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.     

Interpretation of secondary electron images obtained using a low vacuum SEM

Charging of insulators in a variable pressure environment was investigated in the context of secondary electron (SE) image formation. Sample charging and ionized gas molecules present in a low vacuum specimen chamber can give rise to SE image contrast. "Charge-induced" SE contrast reflects lateral variations in the charge state of a sample caused by electron irradiation during and prior to image acquisition. This contrast corresponds to SE emission current alterations produced by sub-surface charge deposited by the electron beam. "Ion-induced" contrast results from spatial inhomogeneities in the extent of SE signal inhibition caused by ions in the gaseous environment of a low vacuum scanning electron microscope (SEM). The inhomogeneities are caused by ion focusing onto regions of a sample that correspond to local minima in the magnitude of the surface potential (generated by sub-surface trapped charge), or topographic asperities. The two types of contrast exhibit characteristic dependencies on microscope operating parameters such as scan speed, beam current, gas pressure, detector bias and working distance. These dependencies, explained in terms of the behavior of the gaseous environment and sample charging, can serve as a basis for a correct interpretation of SE images obtained using a low vacuum SEM.     

Specimen coating for high resolution scanning electron microscopy

Thin conducting films, produced by evaporation or soft vacuum sputtering generally show cracks and grain formation, when examined under high resolution scanning electron microscopy (SEM). These artefacts can obscure surface features of coated specimens or cause confusion in the interpretation of micrographs. No such structures have been observed in films produced by ion beam deposition. Ion beam deposition equipment is described in which a cold cathode saddle field ion source, operating at low pressure (15mPa), produces a 2 mm diameter beam of energetic ions (5 keV) and neutrals. With the beam directed onto a target at 30° to glancing incidence, the sputtered material coats the specimens, which are held in a planetary system for good coverage. Conditions favouring fine grain growth are a high nucleation density and low energy transfer to the substrate by thermal conduction or radiation or by particle or photon radiation. These conditions are satisfied by ion beam deposition but evidently not by evaporation or soft vacuum sputtering. With the specimen stationary, sharp shadowing is obtained because the target acts almost as a point source, because of the small diameter of the beam and because there is little scatter at the operating pressure.     

Specimen charging and detection of signal from non-conductors in a cathode lens-equipped scanning electron microscope

This paper concerns the problems connected with the observation of a nonconductive specimen in a scanning electron microscope (SEM) when incident electrons create a surface charge and a corresponding electric field. The special configuration of the cathode lens enables one to control the landing energy of primary electrons via the specimen bias. In the cathode lens, the accelerating electric field at the surface of the specimen combines itself with that of the surface charge in influencing the trajectories of the signal electrons and hence the detected signal level and the possible recapturing of slow secondaries. Recaptured electrons reduce the ultimate positive surface potential, which arises when working below the higher critical energy of electron impact. Computer simulations of electron trajectories were performed for the typical cathode lens configuration and for a model specimen characterized by emission yields similar to those for glass. The simulations brought an extensive set of data about the trajectories of both secondary and backscattered electrons. Furthermore, the data were processed in order to assess the charge balance between the emitted and recaptured electrons as well as the collection efficiency of the detector. The results include values of the ultimate positive surface potential and the detected signal level, both in dependence on the initial energy of the electron impact and the size of the field of view. Finally, the method for the determination of critical energy is reevaluated. This is based on the measurement of the time dependence of the detected signal.     

Reduction of charging effects using vector scanning in the scanning electron microscope.

We describe a vector scanning system to reduce charging effects during scanning electron microscope (SEM) imaging. The vector scan technique exploits the intrinsic charge decay mechanism of the specimen to improve imaging conditions. We compare SEM images obtained by conventional raster scanning versus vector scanning to demonstrate that vector scanning successfully reduces specimen-charging artifacts.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

The use of charging effects in Al/Al2O3 metal-matrix composites as a contrast mechanism in the SEM

A technique is described to image two phases (alumina and spinel) within a metal-matrix composite which takes advantage of charging effects that occur during examination in an SEM. Microscope and specimen parameters which affect the amount of contrast generated via charging are discussed, and imaging strategies are introduced to optimize the effect. “Model” metal-matrix composite specimens were developed to verify the degree of charging in each phase.     

Collection deficiencies of scanning electron microscopy signal contrasts measured and corrected by differential hysteresis image processing

Limitations of scanning electron microscopy (SEM) image resolution and quality were measured in digital image data and their effect on image contrasts was analyzed and corrected by differential hysteresis (DH) processing. DH processing is a mathematical procedure that utilizes hysteresis properties of intensity variations in the image for a segmentation of differential contrast patterns. These patterns display contrast properties of the data as coherent full-frame images. The contrast segmentation is revertible so that the original image can be restored from the sum of the sequentially extracted DH contrast patterns. DH imaging enhances weak contrast components so that they are more easily recognizable and displays SEM image data free of signal collection efficiency contrasts. Example image data include environmental SEM (ESEM) and SEM images of low and mediumhigh magnifications where collection deficiencies included charging of the specimen surface, obstructions from specimen topography, and uneven signal collection properties of the detector. ESEM low-vacuum image data, which appear to be of high quality, contained local areas of reduced contrasts due to residual surface charging. In such areas, signal contrasts were reduced up to 80%, which suppressed most of the weak short-range contrasts. In low-magnification SEM images, up to 93% of the local high precision contrast was lost from the various adverse effects which diminished the pixel-related contrast resolution of the microscope and resulted in images with low detail. Also, at medium magnification, surface charging effects dramatically reduced the image quality because contrasts resulting from local electron beam/specimen interactions were reduced by as much as 71%. DH imaging restored the local contrast losses by elimination of the collected distorted fraction of signal contrasts and reconstitution of the collected maintained fraction. Restored DH images are of superior quality and enhance the imaging capability of the conventional SEM. DH contrast segmentation provides an improved basis for the measurement of various signal contrast components and detector performances. The DH analysis will ultimately facilitate a precise deduction of specimen properties from extracted contrast patterns.     

Static and dynamic experiments in cryo-electron microscopy: comparative observations using high-vacuum, low-voltage and low-vacuum SEM

We carried out a unique comparative study between three modes of cryo‐scanning electron imaging: high‐vacuum, low‐voltage and low‐vacuum, using ice cream as a model system. Specimens were investigated both with and without a conductive coating (Au/Pd) and at temperatures for which ice either remains fully frozen (< ?110 °C) or undergoes sublimation (?110 to ?90 °C). At high magnification, high‐vacuum imaging of coated specimens gave the best results for ‘static’ specimens (i.e. containing fully frozen ice). Low voltages, such as 1 kV, could be used for imaging uncoated specimens at high vacuum, although slight ‘classical’ charging artefacts remained an issue, and the reduced electron beam penetration tended to decrease the definition between different microstructural features. However, this mode was useful for observing in situ sublimation from uncoated specimens. Low‐vacuum mode, involving small partial pressures of nitrogen gas, was particularly suited to in situ sublimation work: when sublimation was carried out in low vacuum in the absence of an anti‐contaminator plate, sublimation rates were significantly reduced. This is attributed to a small partial pressure of sublimated water vapour remaining near the specimen surface, enhancing thermodynamic stability.     

Simulations of scanning electron microscopy imaging and charging of insulating structures

We present a three‐dimensional simulation of scanning electron microscope (SEM) images and surface charging. First, the field above the sample is calculated using Laplace's equation with the proper boundary conditions; then, the simulation algorithm starts following the electron trajectory outside the sample by using electron ray tracing. When the electron collides with the specimen, the algorithm keeps track of the electron inside the sample by simulating the electron scattering history with a Monte Carlo code. During this phase, secondary and backscattered electrons are emitted to form an image and primary electrons are absorbed; therefore, a charge density is formed in the material. This charge density is used to recalculate the field above and inside the sample by solving the Poisson equation with the proper boundary conditions. Field equation, Monte Carlo scattering simulation, and electron ray tracing are therefore integrated in a self‐consistent fashion to form an algorithm capable of simulating charging and imaging of insulating structures. To maintain generality, this algorithm has been implemented in three dimensions. We shall apply the so‐defined simulation to calculate both the global surface voltage and local microfields induced by the scanning beam. Furthermore, we shall show how charging affects resolution and image formation in general and how its characteristics change when imaging parameters are changed. We shall address magnification, scanning strategy, and applied field. The results, compared with experiments, clearly indicate that charging and the proper boundary conditions must be included in order to simulate images of insulating features. Furthermore, we shall show that a three‐dimensional implementation is mandatory for understanding local field formation.     

Ductility loss of hydrogen-charged and releasing 304L steel

The mechanical properties and fracture behavior of 304L austenitic stainless steel after cathodic hydrogen charging and hydrogen spontaneously releasing are investigated by tensile tests. Flat tensile specimens were cathodic hydrogen charged at various current densities. For each density, two specimens were charged at the same condition. When the charging process completed, one specimen was tensile immediately to fracture and the other was aged to release the hydrogen out of it and then was also tensile to fracture. The resulting tensile properties and micrographs of fracture surfaces of these specimens were evaluated and compared. The results show ductility loss occurred in the hydrogen-charged specimens and the loss increased as the current density increasing. After hydrogen releasing, the specimens recovered a certain extent but not all of its original ductility. Scanning electron microscope (SEM) micrographs of fracture surfaces reveal that irreversible damage had developed in the hydrogen-releasing specimens during the releasing process rather than the charging process. This consequence can be ascribed to the high tensile stress caused by non-uniform hydrogen distribution during hydrogen releasing.     

Consequences of positive ions upon imaging in low vacuum scanning electron microscopy

The effects caused by an excess quantity of ionized gas molecules within the low vacuum, variable pressure and environmental scanning electron microscope (ESEM) are described with reference to mechanisms by which they can influence imaging conditions. These effects can include specimen charging, recombination and development of space charge. They are demonstrated for three different classes of sample: (1) an electrically grounded conductor, (2) an electrically floating conductor, and (3) an electrical insulator. A new device is presented that will aid excess charge removal within the ESEM and help correct for some of these effects, thereby dramatically improving imaging over a wide range of operating conditions and samples. The mechanism of image enhancement is demonstrated with reference to the three classes of sample described above.     

Control of charging in low-voltage SEM

Charging of the specimen under electron beam irradiation is a common problem in scanning electron microscopy (SEM). It results in unstable imaging conditions and a loss in resolution due to defocus of the beam. In addition, it can cause permanent changes in some specimens from translocation of mobile ions under the influence of the induced electrostatic field. To minimize charging and its associated problems, the incident beam energy must be carefully chosen to be the value E₂ at which a dynamic charge balance is obtained. This article presents data on E₂ values for a variety of materials and demonstrates how E₂ is affected by the choice of angle of beam incidence.     

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.     

Charging phenomena in PEEM imaging and spectroscopy

Spectromicroscopy with the imaging technique of X-ray photoelectron emission microscopy (X-PEEM) is a microchemical analytical tool installed in many synchrotron radiation laboratories, and which is finding application in diverse fields of research. The method of sample analysis, X-ray absorption spectroscopy, does not encounter the same problems as X-ray photoemission spectroscopy when sample charging occurs, hence even good insulators may often be analyzed without any apparent artifacts in images or spectra. We show, however, that charging effects cannot be neglected. We model the effect of surface charge formation on the secondary electron yield from uniform samples to demonstrate that surface charge primarily reduces the yield of electrons which may contribute to the detected signal. We illustrate that on non-uniform insulating samples, localized centers of charge may substantially affect microscope imaging and resolution as the electrostatic field close to the surface is distorted. Finally, in certain circumstances non-uniform surface charge may lead to unexpected lineshapes in X-ray absorption spectra causing, in some extreme cases, negative spectra. These negative spectra are explained, and several strategies are reviewed to minimize the impact of sample charging when analyzing poorly conducting samples of any nature.     

Iodine vapor staining for atomic number contrast in backscattered electron and X‐ray imaging

Iodine imparts strong contrast to objects imaged with electrons and X‐rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation‐deposition state‐change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas‐tight container along with a small mass of solid I₂. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin‐embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X‐ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron‐ and photon‐sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.     

Bean-induced loss of organic mass under electronmicroprobe conditions

Techniques for obtaining complementary replicas have already been shown to be valuable in aiding the interpretation of electron micrographs of replicas of specimens prepared by freeze-etching and freeze-fracturing techniques, and in the recognition of artefacts. This paper describes a simple and efficient method of obtaining complementary pairs of replicas of all types of specimen. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end-to-end position using a special pair of forceps. Up to six rivets are placed in a device consisting of a hinged plate held together with clamps against the force of small springs. When the clamps are released, the pair of rivets are separated, fracturing the specimens. The device is easily adapted for use in any type of freeze-etching or high vacuum apparatus. On the example of Saccharomyces cerevisiae the application of the technique to the detection of artefacts in complementary replicas are described. It was shown that the fibrils observed on cross-fractured cell walls are produced by plastic deformation of cell wall components.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.