Helium ion microscopy for low-damage characterization and sub-10 nm nanofabrication

This review introduces the technique of helium ion microscopy along with some unique applications of this technology in the fields of electronics and biology, as performed at the National Institute of Advanced Industrial Science and Technology, Japan, over the last several years. Observations of large-scale integrated circuits, analyses of low-dielectric-constant films with minimal damage, and assessments of copper metal in insulating films are discussed. The special characteristics of this technique are explained, including low-energy input to the material and minimal secondary electron energy resulting from helium ion irradiation. Applications to electronic materials, such as tuning the electrical conductivity of graphene films by helium ion beam irradiation and the formation of nanopore arrays on graphene films with nanometer-scale control, are presented. The use of helium ion microscopy to examine cellular tissues based on the low damage imparted by the ion beam is also evaluated.


Introduction
The technique of helium ion microscope (HIM) remains unfamiliar to many, including those in the scientific community. HIM is based on the projection microscope first proposed by Muller in 1951 [1], in which a rare gas adsorbed on a pointed metal tip is field ionized and accelerated straight ahead, and a magnified image of the metal tip surface is projected onto a fluorescent plate on the atomic scale for imaging. The metal tip in a rare gas atmosphere is used as a filament of the ion source in HIM and is called a gas field ion source (GFIS) microscope. In 1975, Escovitz et al. developed the hydrogen ion transmission microscope [2], while Orloff et al. proposed the present GFIS microscope in 1978 [3]. At the same time, a liquid metal ion source was developed that enabled more stable beam extraction, leading to the research, development, and commercialization of gallium (Ga)-focused ion beam (FIB) microscopes, which are now widely used [4,5]. The success of these instruments subsequently stalled the research and development of GFIS microscopes.
In 2006, more than a decade after Ga FIB microscopy technology reached maturity, HIM instrumentation was commercialized by the Atomic Level Ion Source (ALIS) Corporation, an American venture company [6]. In this system, helium ions drawn from the tip of a sharpened metal ion source in a helium gas atmosphere are focused by a lens system and irradiated to the sample. Using this technique, structures in the near-surface region can be evaluated based on the analysis of secondary electrons (SEs), backscattered ions, ionoluminescence (IL), and other effects generated in the near-surface region. Since 2006, there has been steady progress in the research and development of HIM technology with improvements in the associated instrumentation. Consequently, this technique has found new applications in areas where scanning electron microscopy (SEM) and transmission electron microscopy (TEM) cannot be used or are difficult to use. Wolff et al. previously evaluated the physics of FIB microscopes as a means of better understanding the difference between the effects of ion species (such as gallium, xenon, and helium ions) and those of FIB systems [7]. Hlawacek et al. describe in detail the fundamentals of helium ion microscopy, application on imaging, nanofabrication, and analysis, and theoretical models [8].
The present author introduced HIM to the National Institute of Advanced Industrial Science and Technology (AIST), Japan, for the first time in 2010. This initial work focused on the observation of silicon (Si)-based largescale integrated (LSI) circuit structures and materials as well as copper (Cu)/low dielectric constant (low-k) multilayer interconnect structures. HIM was demonstrated to be a superior technique compared with SEM in terms of performing low-damage observations of low-k film materials with minimal deformation and assessments of Cu metal in insulating films [9]. HIM was found to provide several advantages, such as less charge-up, reduced damage to the sample, and greater depth of focus. The present review is based on the work of the author at AIST and is intended to provide an overview of the HIM system. The studies of low-k materials and Cu metal referenced above are also discussed, and the reasons why such observations are possible with HIM are evaluated. In addition, as a means of showcasing the applications of this technology to the processing of electronics materials and the fabrication of devices, the author describes the tuning of the electrical conductivity of graphene films by helium ion beam irradiation and the formation of nanopore arrays on graphene films. The evaluation of cellular tissues based on the low-damage properties of HIM is also described. The applications of HIM technology to electronic device engineering and life sciences are explained in a broad, generalized manner.

HIM instrumentation
Both Ga FIB microscope and HIM employ essentially the same ion optics, with the major difference being that the ion source is a helium gas field in the latter and liquid Ga metal in the former. Figure 1 provides a diagram of a basic HIM system along with a HIM trimers (beams) image [6,10]. In a HIM system, the source consists of a sharpened needle made of tungsten (which has a high melting point) held at high positive voltage and low temperature of 73 K so as to reduce thermal vibrations in the presence of helium gas of approximately 10 −4 Pa. Figure 1a shows the typical form of the ion source, which is much like an electron emitter in shape. Field ionization allows the creation of a very bright ion beam at the tip of the needle, as described next. A special source formation process creates a tip having just three atoms (the "trimer") at the very apex so that the leading edge is more convex than the remainder of the tip, where the electric field is higher than in the surrounding area. Helium atoms adsorbed on the ion source or in Brownian motion in the vicinity of the source are preferentially ionized at this leading edge in a red dotted circle in Fig. 1a. The electric field can become intense enough over these atoms that gas atoms impinging on the surface are stripped of their Fig. 1 a A diagram of a HIM gas ion source and ion optics and b an image of a helium ion trimer generated by ionization at the tip of the source. One of the three helium ion beams (trimer) drawn from the three atoms at the front edge is selected as the beam for sample irradiation using an aperture and accelerated at 10-35 kV. Figure modified from [6,9] electrons and emitted as ions. Cryogenic temperature at the tip increases the density of available gas atoms. This source geometry creates high brightness emission from the three trimer atoms as shown in Fig. 1b, which is a schematic of the helium ion launch site and of the single atom from which the scanning probe is formed. Selecting one of these atoms as the source delivers a beam with a source size below an Angstrom and a brightness at voltage exceeding 5 × 10 9 A cm −1 sr −1 , an order of magnitude beyond even a cold field electron emitter. The ion beam is then transmitted through a lens electrostatic ion optical column. This has a traditional optical configuration and is run in a non-crossover mode with pre-lens deflection. The beam landing energy can be varied from 10 to 35 keV, in which beam currents from 0.1 pA to tens of pA are used. The ion optical column has produced a focused probe with a spot size of about 0.35 nm. This number is predicted by optical calculations and verified experimentally [8]. Finally, the small source size allows the column to be operated with less demagnification and thus a small beam convergence angle. This leads to a long depth of field for images captured with the microscope up to several times of what is obtained in an SEM. SEs, backscattered helium ions, and luminescence generated in the surface near region are monitored as a means of observing the sample. A very high spatial resolution of 0.35 nm is currently available in conjunction with the edge contrast evaluation of graphite samples. Interestingly, the ion irradiation itself can be used to modify the physical properties of the specimen with nanometerscale precision and to perform etching.
Thus, because the HIM beam originates from an atomic scale point source, extremely high resolution can be obtained. Even so, it is extremely important to keep the leading edge of the ion source clean and to maintain a constant tip morphology in order to generate a stable beam with minimal fluctuations over time, and further improvements are required with regard to this aspect of the technology.

Observations of low-k interconnect dielectric films
In the manufacturing of Si-based LSI circuits, the thermal damage caused by electron beam irradiation during high-magnification observations by SEM may lead to shrinkage deformation. This can occur during stages such as the observations of the photoresist after lithography process, the low-k inter-wiring insulating films as part of the multilayer interconnect process after the dry etching, and so on. This effect is especially noticeable in case of fine patterns with a pitch of 100 nm or less. As an example, when evaluating the shape of a low-k inter-wiring insulating film pattern using SEM, it can be difficult to determine the exact shape and dimensions of the pattern because, depending on the material used, large deformations may occur even during a single observation involving an electron beam. Figure 2a shows structure of a low-k inter-wiring insulating film pattern observed by HIM before the embedding of copper interconnects [9]. The wiring pitch in this pattern was 140 nm (70 nm/70 nm line and space), and the film comprised a stacked structure made of materials having dielectric constants of 3.1 and 2.4. Note that, prior to the observation of this specimen, a conductive coating to prevent charging was not applied, as would be required for SEM observation. In the case that charging would be expected to adversely affect the imaging process, interleaved irradiation of electron beam by an Fig. 2 a A diagram of a low-k interlayer insulating film pattern before embedding copper interconnects and b a HIM image of the actual structure. Note that a conductive thin film to prevent charge-up did not have to be applied to this specimen. Figure modified from [9] electron gun and He ion beam to the sample is used to prevent charging. Figure 2b presents a HIM image acquired at 0.1 pA and 32 kV without any electron irradiation. This image indicates that there was no significant deformation. In addition, fine undulations and irregularities (with sizes of less than 10 nm) on the side walls, bottom, and surface of the film are clearly visible in the depth direction. Note that, during these observations, the depth of focus achieved using HIM was approximately six times that possible with SEM.
Interestingly, this work produced a seemingly contradictory result, in that irradiation with helium ions (which have a mass approximately 8000 times that of electrons) resulted in less deformation of the low-k film compared with SEM analyses. Several reasons can be proposed to explain this result. Figure 3 plots the irradiation power densities for helium ions and electrons per unit volume as functions of the irradiation beam current when observing samples by HIM (40 kV) and SEM (1 kV). It is evident that, when employing conditions related to HIM (such as 0.1 pA and 40 kV) and conventional SEM (10 pA, 1 kV), the power densities received by the sample were approximately 7 × 10 6 W/cm 2 and 2 × 10 9 W/cm 2 , respectively. Thus, the value associated with HIM observations was more than two orders of magnitude smaller than that for SEM, suggesting that the temperature rise in the region of the low-k film exposed to the beam would be lower, and thermal deformation would therefore be reduced. In addition, because the beam irradiation during HIM imaging is much lower than that during SEM imaging, the amount of electron beam irradiation required for neutralization is also very low. Therefore, even if neutralization is necessary, a low-k film will not shrink under the influence of the neutralization beam.
From the above observation experiments, it can be assumed that helium ions produce SEs more efficiently than electrons, and that the HIM beam current required to image a sample with an elemental composition consisting of carbon (C), hydrogen (H), oxygen (O), and silicon (Si) is less than the current required for an SEM. Therefore, in addition to the materials mentioned above, HIM is extremely advantageous when observing and evaluating samples that are susceptible to thermal loading, such as photoresists in LSI circuit fabrication processes and biological tissues, because the extent of thermal deformation is less than when using SEM.

Observation of a Cu interconnect embedded in an interlayer dielectric
Figure 4a provides a cross-sectional diagram of the sample that was evaluated in these trials [9]. The diameter of each multilayer interconnect through hole (labeled "Vias" in the figure) between the top and bottom multilayer interconnect was approximately 1 μm. There was no upper layer interconnect (M2) but rather only a lower layer interconnect (M1(Cu)), and the sample was observed using a helium ion beam applied from above, as indicated by the arrow in Fig. 4a. The resulting HIM image is shown in Fig. 4b. The Cu surfaces in the pores (that is, the regions labeled as "Vias") are visible, as are the M1 (Cu) interconnect under the 30-nm-thick SiO 2 cap and the 100-nm-thick low-k film (totaling a 130 nm insulation layer). A white-to-black crystal contrast is clearly observed in the Cu metal regions, which can be In the region associated with HIM observations (such as at 0.1 pA), the power density is more than two orders of magnitude lower than in the regions associated with SEM observations (such as at 10 pA). Figure modified from [9] attributed to channeling of helium ions in conjunction with specific grain orientations.
In the case that the contrast image is based on surface potential, internal potential, or capacitive coupling, as has been reported with regard to conventional SEM observations [11], the interconnect will not exhibit crystal contrast due to channeling, and so, a different image will be obtained. The details of this difference require further study, but a qualitative assessment is possible. In these imaging experiments, a 32 keV helium ion beam passed through the insulating film and impacted the Cu metal surface, which would be expected to generate a large amount of SEs with a peak in the energy distribution at approximately 1 eV [12,13]. These SEs would have diffused into the insulating film, which had a band gap of 5 to 9 eV [14], and would then have been ejected from the surface of the film. In contrast, He ions backscattered from the Cu metal surface could have generated SEs near the insulating film surface. Considering the backscatter of helium ions and the performance of the SE detector in the HIM system, the former mechanism is thought to have occurred, although further research based on experimentation and theory is necessary.

Modifying the electrical conductivity of graphene films
In 2009, Lemme [15] and Bell [16] reported the etching of graphene by HIM, based on the helium ion beam irradiation associated with this technique. And several groups studied the nanofabrication using HIM [17][18][19]. In addition, the author's group proposed that the charge transport state in graphene could be tuned using the spatial localization of charge resulting from charge scattering near the Dirac point [20,21]. The aim was to accomplish this effect by introducing and controlling crystal defects in graphene while applying a helium ion beam. The formation of graphene nanoribbons having a width of several nanometers, control of the electrical conductivity of graphene films, and the fabrication of electronic devices has also all been realized by using helium ions [21][22][23][24]. The energy range over which electrical conductivity is suppressed near the Dirac point is referred to as the transport gap. Here, the tuning of electrical conductivity based on the random introduction of atomic-scale crystal defects at suitable concentrations using the helium ion irradiation that occurs with HIM is described [25][26][27][28][29]. Figure 5 shows the results obtained from scanning probe microscopy observations of graphene films on a Si/SiO 2 substrate irradiated with helium ions at densities in the range of 5.2 × 10 15 to 2.0 × 10 16 ions/cm 2 with an acceleration voltage of 30 kV [25][26][27]. In Fig. 5a, the irradiated area is slightly darker than the surrounding area, which can be attributed to the change in physical properties of the graphene film itself, to the charge-up effect, and possibly also to carbon deposition during irradiation. Figure 5b shows the surface roughness as determined using atomic force microscopy (AFM). A line profile of this image is also presented (in red) in Fig. 5d. It is apparent that the irradiated area was approximately 2 nm deeper Fig. 4 a Cross-sectional diagram of the Cu metal/low-k film structure that was observed and b a HIM image of the buried M1 metal acquired from the top of the sample through the 130-nm-thick insulating film. The crystal contrast in the Cu interconnect region can likely be ascribed to channeling of helium ions. Figure modified from [9] than the unirradiated regions. The reason for this phenomenon is currently unknown, and further investigation is necessary. When evaluating a given thin film with a given structure using capacitance microscopy, the capacitance between the probe and the film changes as the probe moves from the conductive to the insulating part of the film, and this principle can be used to determine the conductivity over minute areas. The dark-brown area in Fig. 5c corresponds to the region over which a change was observed by capacitance microscopy. The blue plot in Fig. 5d indicates that a decrease of approximately 8 mV can be seen only in the 2.0 × 10 16 ions/cm 2 irradiation region, demonstrating that the monolayer graphene became insulating.
The crystal defect density in the film was calculated from the density of incident ions during the trials described above [16,30] and was determined to equal approximately 0.1 to 2% of the total number of carbon atoms, while irradiation at 2.0 × 10 16 ions/cm 2 formed approximately 2% defects. These defects would be expected to localize carriers in the monolayer graphene and so increase the resistance of the material and transform the graphene into an insulator. Figure 6 shows   6 Raman spectra of graphene films subjected to varying helium ion doses. Although the intensity of the D band was increased by helium ion irradiation, there was no significant change in the G band, indicating that defects were generated, while the honeycomb framework was well maintained. Figure modified from [22] Raman spectra acquired from graphene films after irradiation with varying helium ion doses [22]. The G-band in these spectra at approximately 1580 cm −1 originated from the in-plane motions of carbon atoms and so could be used to ascertain the number of layers because its intensity was affected by strain. In contrast, the D-band (1270-1450 cm −1 ) was related to structural disorder and crystal defects and could be used to evaluate such defects. The D-band intensity evidently increased as the ion irradiation was increased, while the G-band intensity changed only slightly. These data suggest that crystal defects were introduced into the graphene film by ion irradiation, but the honeycomb framework crystal structure was maintained.
The effects of the helium ion dose on the current-voltage characteristics of single-layer graphene deposited on a SiO 2 film were investigated, and Fig. 7 a and b presents a diagram and HIM image of the prototype device, respectively [22]. In this device, the source and drain electrodes associated with Ti/Au multilayers (the yellow region in (a)) were formed at both ends of the unirradiated graphene film within a 50-nm-wide (W irr ) and 30-nm-long (L irr ) channel by irradiation with helium ions at a density of 2.2 × 10 15 ions/cm 2 to 1.3 × 10 16 ions/cm 2 (giving a crystal defect concentration of 0.2 to 1.3%). A relatively high dose of 1.3 × 10 16 ions/cm 2 was applied over a length of 200 nm (the dark insulating area in (a) and the dark area in (b)). This zone served to electrically isolate the graphene in the source and drain regions, such that the source-drain conductance was primarily determined by the 50 nm × 30 nm region seen in the center of the HIM image in Fig. 7b. Figure 7c presents current-voltage data obtained between the source and drain at room temperature. The current is seen to have decreased rapidly with increasing irradiation dose, and, at a dose of 13.1 × 10 15 ions/cm 2 , the graphene was almost an insulator.
These results confirmed the feasibility of fabricating nanoscale devices by introducing crystal defects in a controlled manner and tuning the conductivity by changing the electronic state using helium ion beam irradiation in conjunction with HIM. Conductivity tuning using crystal defect formation by helium ion beam irradiation has also been studied in other two-dimensional thin films of MoS 2 [31] and high-temperature superconducting thin films of YBa 2 Cu 3 O 7 [32].

Nanopore microfabrication in suspended graphene films
There has recently been remarkable progress in the miniaturization and speed of nanoelectromechanical systems (NEMS) based on the application of microfabrication technology to the fabrication of Si-based LSI circuits. High-resonance-frequency oscillators [33] and sensor elements [34] have been reported. In addition, high-performance switching characteristics have been improved, and multifunctional elements have been developed by incorporating NEMS structures into Si devices [35]. Novel phonon phenomena in nanostructured materials Fig. 7 Assessment of the effect of helium ion dose on the current-voltage characteristics of a single layer of graphene. a A schematic diagram of the test device, b a HIM image, and c a current-voltage plot. These data indicate that the graphene was transformed from the metal to the insulator by ion irradiation. Figure modified from [22] have also been studied by fabricating NEMS structures with quantum dots and nanopores on nanoscale oscillators [36][37][38]. Graphene can be used in NEMS structures and has superior mechanical properties compared with Si, including an order of magnitude larger than Young's modulus and high resonance frequency modulation [39][40][41]. The author's group has fabricated nanoribbons having widths of several nanometers [42,43] and nanopore arrays with diameters of several nanometers and pitch values of less than 20 nm [44,45] on suspended graphene films using HIM. Phonon engineering has been used to quantize the energy of acoustic phonons confined at both fixed ends of such specimens. Although NEMS devices are becoming increasingly miniaturized, it is difficult to fabricate these units on the scale of 10 nm or less using conventional Ga FIB technology because the beam diameter of several nanometers is too large and the fabrication-induced damage is too severe. Conversely, the beam diameter associated with an HIM instrument is less than 1 nm, and so, devices with features of 10 nm or smaller can be fabricated. In addition, since the atomic mass of helium is only approximately 1/18 that of gallium, the sputtering rate for carbon is reduced in the same proportion, while the graphene film is so thin that it does not interfere with the etching process. Figure 8 shows an example of the fabrication of a pore array on a graphene film supported by metal electrodes at both ends based on exposure to a helium ion dose of 6.35 × 10 5 per micropore (6.5 × 10 19 ions/cm 2 in total) using HIM with an acceleration voltage of 30 kV [45]. The contrast in the image confirms that it was possible to fabricate a fine pore array with a diameter of approximately 4.2 nm and a pitch on the order of 9.0 nm. Interestingly, varying the pore size and the stress to which the suspended graphene was subjected was found to significantly deform the graphene. The extent of this deformation was determined by the ion dose and irradiation pitch, and so, both the pore size and alignment had to be optimized to suppress stress generation. A graphene device with a pore array having an 18-nm pitch and 4-nm-diameter pores exhibited reproducible electrical properties and a transport gap of approximately 450 meV at room temperature [45]. This gap is thought to have resulted from the disordered atomic arrangement at the processing edge and crystal defects that occurred during nanopore processing, although further study is needed.

Application to biological samples
Thus far, this review has described the application of HIM technology to materials and processes for fabricating electronic devices. However, as noted in Section 3.1, HIM is also very effective as a means for observing materials that are easily deformed by thermal loading. As such, this technology allows the assessment of biological specimens at high resolutions, which would normally require a high-energy-density beam. In fact, as a rough estimate, approximately one-third of all the applied research based on HIM worldwide has been related to the observation of biological samples [46]. There have been many reports concerning the analysis of such samples that were very challenging to examine using SEM or TEM but that were easily observed within a short time span using HIM. In Fig. 8 a A HIM image of a nanopore array (indicated within the square) drilled using a dose of 6.35 × 10 5 helium ions per hole and b the profile along the yellow line in (a). These data indicate that a nanopore array with a diameter and pitch of approximately 4.2 nm and 9.0 nm, respectively, could be fabricated. Figure modified from [45] this section, examples of observations and evaluations of cellular tissues are briefly presented, without discussing cytology. Figure 9 provides an image showing the microstructure of a cell surface including the cell membrane caveolae microdomains. The caveolae are flask-shaped depressions with diameters of 50-100 nm on the surface of the cell membrane having oligomeric backbones. These are parts of cholesterol-and sphingolipid-rich rafts that take in substances such as proteins necessary for cell metabolism and play an important role in signal transduction [47]. This sample was prepared using a waterbased freeze-drying method [48]. In the case of the conventional organic solvent freeze-drying method, the cell membrane structure is destroyed by the organic solvent, which is avoided when using water. Caveolae with diameters of 20-50 nm on the membrane surface could be observed with high resolution and significant depth of focus. The microscopic morphologies of caveolae are generally observed by cross-sectional TEM [49] or SEM after employing freeze-fragmentation or replica methods to prepare specimens [50]. The clear image of the outer surface of the cell membrane generated using HIM as shown here was an unprecedented result [51]. The upper right part of Fig. 9 also shows a number of smaller pits that might be lipid rafts, as previously reported by Schurmann et al. [52].
Here  [53] after electron staining with heavy metal compounds such as uranium (https:// www. hitac hi-hight ech. com/ jp/ scien ce/ techn ical/ tech/ micro scopes/ elect ron-micro scope/ techn ique/ chapt er1_5. html). The glomerulus has the function of filtering waste products and salt from blood in the kidney and discharging these substances in urine. It is composed of thin capillaries entwined like yarn and has a size of approximately 0.1 to 0.2 mm. Because red blood cells and proteins are not filtered out, clean blood leaves the kidneys based on this system (https:// www. jsn. or. jp/ global/ gener al/_ 3227. php). The HIM image shown in Fig. 10a has higher contrast than the TEM image in Fig. 10b. The dark blood plasma (BP) in the glomerular capillaries is seen to uniformly surround the bright red blood cells (R), while outside the capillaries, cells are observed to have nuclei (N) with surrounding light density areas (chromatin) and round areas (nucleoli). At higher magnification, mitochondria, endoplasmic reticulum (indicated by the white arrowheads), and vacuoles (indicated by the white arrows) can be distinguished in the cytoplasm (CP). Although these tissues are primarily composed of hydrogen, oxygen, carbon, and nitrogen atoms, HIM allowed the surface structures to be observed with a high degree of contrast without the pretreatment required for SEM observations. Note also that the contrast between the HIM and TEM images is slightly reversed for the red blood cells and cytoplasm. This contrast reversal might be related to variations in electron density between the two techniques, because a higher electron density will result in more beam scattering in the case of the TEM technique, which in turn will generate dark zones in bright-field (BF) images. The same high-density regions will, however, emit more SEs during non-transmissive HIM SE imaging and so will appear brighter. While the interpretation of such images will require much discussion in the future, the application of HIM to the observation of cellular tissues is obviously able to provide new insights when used in conjunction with TEM.
Finally, it is of interest to examine results of observation and evaluation using the IL method in conjunction with helium ion irradiation [54,55]. Specifically, here the application of this technique to the analysis of cell tissue is examined. Figure 11 presents an SE image, an IL image, and superimposed SE and IL images of a sample of renal fibroblast antigen determinants labeled with zinc oxide (ZnO) nanoparticles (several nanometers in diameter), as obtained using HIM. Antigenic determinants are portions of antibodies that recognize and bind to antigens, such as pathogenic microorganisms, and consist of sequences of several amino acids and monosaccharides. Similar to the cathodoluminescence generated by electron beam irradiation during SEM, luminescence can be generated by helium ion irradiation. The spatial distribution of antigenic determinants can be evaluated simultaneously with high-resolution observations. In the case of  [53] the images shown here, a resolution of about 2 nm was obtained [53].
As described above, HIM observations do not require the complicated and time-consuming pretreatments such as electron staining that are necessary prior to TEM. This allows rapid, high-contrast analysis of the fine morphology of cellular tissues, and information of the same or better quality that can be generated using TEM with electronic staining can thus be obtained quickly and easily. Therefore, this technique is very useful for the observation of biological tissues. Even so, it should be noted that the image contrast will be reversed between HIM and TEM images, and so, the interpretation of such images should be carefully considered.

Conclusion
This paper described the application of HIM technology to the observation of Cu/low-k interconnects during the fabrication of Si-based LSI circuits, the control of the electrical conductivity of graphene films, and the analysis of biological tissues. The effectiveness of HIM in performing tasks that are impossible or difficult to accomplish using conventional SEM and TEM techniques was highlighted. It should be noted that many physicochemical phenomena are affected by HIM parameters such as beam current, beam residence time, and irradiation pitch, although these factors were not discussed in this paper. Such phenomena are related to interactions between a helium ion beam focused to a diameter of less than 1 nm and the irradiated sample being evaluated or processed. Therefore, it is necessary to further investigate the future applications of this technique.
New and unique applications of HIM technology should be explored in the future based on collaborations between those working in various fields, such as materials technology, device fabrication and life sciences, and charged beam and equipment technologies. The HIM instrumentation at AIST is operated as a joint use system, and it is the hope of the author that this facility will contribute to the development of HIM and GFIS microscopy techniques both in Japan and throughout the world, through joint research and general use.