The role of helium ion microscopy in the characterisation of complex three-dimensional nanostructures

This work addresses two major issues relating to Helium Ion Microscopy (HeIM). First we show that HeIM is capable of solving the interpretation difficulties that arise when complex three-dimensional structures are imaged using traditional high lateral resolution techniques which are transmission based, such as scanning transmission electron microscopy (STEM). Secondly we use a nano-composite coating consisting of amorphous carbon embedded in chromium rich matrix to estimate the mean escape depth for amorphous carbon for secondary electrons generated by helium ion impact as a measure of HeIM depth resolution.     

Effect of gallium focused ion beam milling on preparation of aluminium thin foils

Focussed ion beam milling has greatly extended the utility of the atom probe and transmission electron microscope because it enables sample preparation with a level of dimensional control never before possible. Using focussed ion beam it is possible to extract the samples from desired and very specific locations. The artefacts associated with this sample preparation method must also be fully understood. In this work, issues specifically relevant to the focussed ion beam milling of aluminium alloys are presented. After using the focussed ion beam as a sample preparation technique it is evident that gallium will concentrate in three areas of the sample: on the surface, on grain boundaries and at interphase boundaries. This work also shows that low-energy Ar ion nanomilling is potentially quite effective for removing gallium implantation layers and gallium from the internal surfaces of aluminium thin foils.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

Minimization of focused ion beam damage in nanostructured polymer thin films

Focused ion beam (FIB) instruments have proven to be an invaluable tool for transmission electron microscopy (TEM) sample preparation. FIBs enable relatively easy and site-specific cross-sectioning of different classes of materials. However, damage mechanisms due to ion bombardment and possible beam heating effects in materials limit the usefulness of FIBs. Materials with adequate heat conductivity do not suffer from beam heating during FIB preparation, and artifacts in materials such as metals and ceramics are primarily limited to defect generation and Ga implantation. However, in materials such as polymers or biological structures, where heat conductivity is low, beam heating can also be a problem. In order to examine FIB damage in polymers we have undertaken a systematic study by exposing sections of a PS-b-PMMA block copolymer to the ion beam at varying beam currents and sample temperatures. The sections were then examined by TEM and scanning electron microscopy (SEM) and analyzed using electron energy loss spectroscopy (EELS). Our empirical results show beam heating in polymers due to FIB preparation can be limited by maintaining a low beam current (≤100 pA) during milling.     

Objective comparison of scanning ion and scanning electron microscope images

Common and different aspects of scanning electron microscope (SEM) and scanning ion microscope (SIM) images are discussed from a viewpoint of interaction between ion or electron beams and specimens. The SIM images [mostly using 30 keV Ga focused ion beam (FIB)] are sensitive to the sample surface as well as to low-voltage SEM images. Reasons for the SIM images as follows: (1) no backscattered-electron excitation; (2) low yields of backscattered ions; and (3) short ion ranges of 20–40nm, being of the same order of escape depth of secondary electrons (SE) [=(3–5) times the SE mean free path]. Beam charging, channeling, contamination, and surface sputtering are also commented upon.     

Imaging and nanofabrication with the helium ion microscope of the Van Leeuwenhoek Laboratory in Delft

Although helium ion microscopy (HIM) was introduced only a few years ago, many new application fields are emerging. The connecting factor between these novel applications is the unique interaction of the primary helium ion beam with the sample material at and just below its surface. In particular, the HIM secondary electron signal stems from an area that is extremely well localized around the point of incidence of the primary beam. This makes the HIM well suited for both high-resolution imaging and high-resolution nanofabrication. Another advantage in nanofabrication is the low ion backscattering fraction, which leads to a weak proximity effect. The subnanometer probe size and the unique beam-materials interactions have opened new areas of research. This review presents a selection of studies conducted on a single instrument. The selection encompasses applications ranging from imaging to nanofabrication and from fundamental academic research to applied industrial developments.     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

Read-out of soft X-ray contact microscopy microradiographs by focused ion beam/scanning electron microscope

A novel focused ion beam-based technique is presented for the read-out of microradiographs of Caenorhabditis elegans nematodes generated by soft x-ray contact microscopy (SXCM). In previous studies, the read-out was performed by atomic force microscopy (AFM), but in our work SXCM microradiographs were imaged by scanning ion microscopy (SIM) in a focused ion beam/scanning electron microscope (FIB/SEM). It allows an ad libitum selection of a sample region for gross morphologic to nanometric investigations, with a sequence of imaging and cutting. The FIB/SEM is less sensitive to height variation of the relief, and sectioning makes it possible to analyse the sample further. The SXCM can be coupled to SIM in a more efficient and faster way than to AFM. Scanning ion microscopy is the method of choice for the read-out of microradiographs of small multicellular organisms.     

Ion source with longitudinal ionization of a molecular beam by an electron beam in a magnetic field

A high-efficiency ion source for a mass-spectrometer’s detector of molecular beams and their scattering products is described. The ion source is designed according to a scheme of impact ionization of a beam particle by a longitudinal electron beam in a magnetic field with a strength of up to 130 mT. The design of the source developed is very flexible and has no limitations for use in any experiments with molecular beams. An ionization efficiency of particles of an atomic helium beam of 10^?3 ions/atom has been achieved. The useful signal-to-background ratio in the detector’s chamber is 3 × 10⁴ during detection of ions with mass-to-charge ratio m/q = 4 amu.     

Mass analyzer using electron-beam-guiding type ion source

A mass analyzer having a scanning electron microscopy (SEM) mechanism and using electron beam bombardment vaporization ion source have been submitted. Secondary ions from the surface trapped in the potential well formed by the incident electron beam can be guided into the slit of mass analyzer through the center of Ta pipe cathode. Efficiency of the collected ions to the evaporated molecules and a qualitative mass analysis with a pressed block of powdered NaCl have been examined. When subsidiary electrodes added above efficiency showed 6.4x10(-4). A sample of 10-mm-thick NaCl has been analyzed along depth direction to the bottom with 0.02 mA at 5 kV in 16 min.     

Controlling FIB‐SBEM slice thickness by monitoring the transmitted ion beam

Serial block‐face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack‐acquisition time to several days. Here, we describe a method for controlling the ion‐beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted‐beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion‐mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in‐column detector is explored.     

Three-colour x-ray mapping using digital storage

Computer-controlled scanning of a scanning electron microscope (SEM) fitted with an energy-dispersive x-ray analyser has been carried out. This has been achieved by building an SEM interface module which controls the scanning of the electron beam and also provides a communication link between the x-ray analyser and an enhanced BBC micro. The digitised x-ray data pulses can be collected from three elements simultaneously to produce colour-coded x-ray distribution maps. Some such maps from two different specimens are presented and an effect of x-ray shadowing is clearly demonstrated. A comparison between images acquired using a frame size with 256 × 256 pixels and those with 128 × 128 pixels is made.     

How to obtain and use X-Ray projection microscopy in the SEM

The primary electron beam of an SEM is used to generate a point source of x-rays, permitting x-ray microscopy within the SEM. The practical device described fits into the sample chamber of an SEM. Exact and reproducible adjustabilities of the sample support and the film holder allow two x-ray micrographs, e.g. stereo-micrographs, to be taken in one run without repressurization of the specimen chamber. By comparing x-ray and SEM micrographs of the same sample in the identical position it is possible to get additional information concerning surface morphology and inner structure of a specimen.     

A surface work function measurement technique utilizing constant deflected grazing electron trajectories: oxygen uptake on Cu(001)

We report on the application of a novel nondestructive in-vacuum technique for relative work function measurements, employing a grazing incidence electron deflection above a sample with a planar surface. Two deflected electron beam detectors are used as a position sensitive detector to control feedback to the sample potential as the sample work function changes. With feedback the sample potential exactly follows the surface sample-size averaged work function variation, so that the deflected beam trajectory remains stable. We also discuss methods to optimize the initial electron trajectories for this method, so as to minimize unwanted effects such as from uncontrolled external magnetic fields. As the electron beam does not impinge on the surface in this new technique electron induced desorption, ionization, dissociation, and/or decomposition is not induced at the interface. Importantly also the technique allows for free access to the surfaces enabling simultaneous deposition/evaporation and/or application of other surface characterization methods. We demonstrate its application in concurrent measurements of helium atom reflectivity and work function changes taking place during molecular oxygen exposure of a Cu(001) surface. A work function measurement sensitivity and stability is demonstrated at ～10?mV at a sampling rate of 1 Hz and after application of an ～7?s smoothing routine. In comparison to the helium atom reflectivity measurements, the work function measurements are more sensitive to the initial O uptake, and less so to the final coverage variations and possible surface reordering at higher O coverages.     

Examples of image processing using a computer controlled SEM

Several examples of the use of digital image processing on SEM images are presented. The emphasis is on image enhancement as opposed to pattern recognition. Examples given include non-linear histogram modification, noise filtering, and frame by frame subtraction. Image processing is done on digitally stored images obtained from a modified SEM. The SEM has been modified to allow minicomputer control of the electron/CRT beam position and blanking. Digitization of the sample signals, such as secondary electron, backscattered electron, and absorbed current is done using a 5 MHz voltage to frequency converter and a 100 KHz timer/sealer combination. Software for image storage and manipulation is also described.     

Ultrastructural comparison of ion beam and radiofrequency plasma etching effects on biological tissue sections

Three dry etching techniques (Ar+ ion beam, O₂+ ion beam, O₂ radiofrequency electrodeless discharge) were compared with respect to preferential etching and damage to the ultrastructure of glutaraldehyde-fixed Epon-embedded frog skeletal muscle sections. SEM and TEM studies were performed on both unstained and stained (osmium tetroxide, uranyl acetate) sections. Etching effects were observed to differ for the various ion beam or plasma etching techniques. Whereas selective retention of electron dense structures (e.g. Z lines, nuclear heterochromatin) was observed for oxygen plasma etching, preferential etching of these components was observed using O₂+ ion beam bombardment. Selectively etched Z lines and etch-resistant nucleoli were observed for both reactive (O₂+) and inert (Ar+) ion beam sputtering after sufficiently high ion doses. The above suggest that selective etching under keV ion beam irradiation is related more to physical sputtering processes (momentum transfer) than to the chemical reactivity of the incident ion. Heavy metal post-fixation and staining had no qualitative effect on the nature of the selective etching phenomena. The above findings are significant in that they potentially influence both electron and ion microprobe measurements of etched biological specimens.     

FIB/SEM characterization of carbon-based fibers

The aim of this paper is to show how a focused ion beam combined with a scanning electron microscope (FIB/SEM machine) can be adopted to characterize composite fibers with different electrical behavior and to gain information about their production and modification. This comparative morphology investigation is carried out on polyacrylonitrile (PAN) carbon fibers and their chemical precursor (the oxidized PAN or oxypan) which has different electrical properties. Fibers are imaged by electron and ion beams and sectioned by the focused ion beam (FIB). A sample of oxypan fibers processed by a radio frequency (RF) plasma is also investigated and the role of the conductive carbon layer around their unmodified, insulating bulk is discussed. A suitable developed edge detection technique (EDT) on electron, ion images, and after the FIB sectioning, provides quantitative information about the thickness of the created layer.     

Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging

Tomography in a focused ion beam (FIB) scanning electron microscope (SEM) is a powerful method for the characterization of three-dimensional micro- and nanostructures. Although this technique can be routinely applied to conducting materials, FIB–SEM tomography of many insulators, including biological, geological and ceramic samples, is often more difficult because of charging effects that disturb the serial sectioning using the ion beam or the imaging using the electron beam. Here, we show that automatic tomography of biological and geological samples can be achieved by serial sectioning with a focused ion beam and block-face imaging using low-kV backscattered electrons. In addition, a new ion milling geometry is used that reduces the effects of intensity gradients that are inherent in conventional geometry used for FIB–SEM tomography.     

Secondary electron imaging as an aid to STEM microanalysis

Secondary electron (SEM mode) imaging in a scanning transmission electron microscope (STEM) has been utilized as an aid to the STEM microanalysis of second phase particles in an Alloy 800 foil by identifying particles at the foil surface facing the incident beam and energy dispersive X-ray detector. Such particles were optimally situated for microanalysis because possible deleterious effects from beam spreading and matrix absorption were eliminated. This was demonstrated by orienting the sample to analyze the particles on the surface of the foil facing away from the beam and X-ray detector, and subsequently, for comparison, reorienting the sample to reanalyze the particles with the foil surface facing beam and detector. Theoretical calculations of the magnitude of the relative change in particle X-ray signal between the two orientations were in good agreement with the experimental observations. SEM imaging also provided surface reference points for parallax-effect measurements of local foil thickness and particle depth within the foil.