Centre For Ion Beam Applications Research Articles

Facilitating focussed proton radiation of single cells by automated cell targeting and novel design of live cell chamber

Targeted radiation of single cells with focussed sub-micron ion beams enables unique investigations in advanced high resolution radiobiology research. Since 2018, the Centre for Ion Beam Applications (CIBA) has operated a dedicated live-cell beam line to study radiobiology on single cells. Recently, we have upgraded this radiobiology beam line with enhanced precision in dose delivery and beam alignment by installing a beam blanking system and stage-mounted exit nozzle, respectively. We have increased efficiency of cell targeting through process automation, combined with exploiting a machine-learning algorithm, Cellpose, for rapid cell recognition to produce a fully automated cell targeting functionality with accurate cell identification. Concordant with further streamlining of our experimental workflows, here we describe the “cell slide”, our novel live-cell chamber assembly, which simplifies and drastically improves throughput and turn-around time in live-cell experiments. Cell slide has been specifically developed to support live-cell radiobiology applications on microbeam platforms.

An optimized 19F (p, αγ)16O elemental mapping facility

A new target stage that allows a close positioning of a large 5in × 5in NaI(Tl) detector was implemented at the 45° beam-line end-station at the Centre for Ion Beam Applications (CIBA). Here we present preliminary results demonstrating the improvement in the F detection limit available in the new geometry. The reaction 19F(p, αγ)16O was used to map fluorine in untreated and fluorine/laser treated areas on human tooth samples.Proton Induced Gamma-Ray Emission (PIGE) was used in combination with Proton Induced X-Ray Emission (PIXE) and Rutherford Backscattering Spectrometry (RBS) for quantitative elemental mapping of fluorine. A 2 MeV proton beam focused to 5~10 μm spot size with a beam current of 0.5–1 nA was used along with a new in-vacuum piezo-driven three-axis target-stage at the nuclear microscopy beamline end-station. The new stage, which replaces the manual target positioning system, optimizes the solid angle subtended by factor of 13.

The radiobiology beam line facility at the Centre for Ion Beam Applications, National University of Singapore

A new beam line facility dedicated to investigations into the radiobiology of biological single cells has been constructed at the Centre for Ion Beam Applications, National University of Singapore. This facility, which has a horizontal layout, has some novel features including post lens magnetic ion deflection and scanning and provision for a diamond exit window and confocal fluorescence microscope for high performance online investigations.The radiobiology facility uses a system of Oxford Microbeam compact magnetic quadrupole lenses enabling high demagnifications (up to 123 × 51) at the cell plane, and a relatively short lens to cell distances (22.5 cm). Beam optics and SRIM calculations have indicated a theoretical design performance of up to 450 nm for the proton spot size at the cell surface.To measure the spatial resolution of the proton beam at the cell position, we positioned a gold calibration grid inside the external cell chamber at a position normally occupied by cells. Preliminary results were obtained by scanning a 2 MeV focused beam over a grid and using a surface barrier detector to construct a STIM image of the grid we have measured an in-air spot size of sub-500 nm at the cell position.In this paper, the design of the new radiobiology beam line, preliminary results of resolution tests, as well as presenting first investigations into the damage caused by focused proton beam irradiation of human liver cancer cells by detecting DNA damage markers gamma-H2AX and 53BP1.

Analytical possibilities of highly focused ion beams in biomedical field

At the Centre for Ion Beam Applications (CIBA), a 3.5MV HVEE Singletron™ accelerator serves to provide MeV ion beams (mostly protons or He+) to six state-of-the-art beam lines, four of which are equipped with Oxford triplet magnetic quadrupole lens systems. This facility is used for a wide range of research projects, many of which are in the field of biomedicine. Here we presented a discussion of currently ongoing biomedical work carried out using two beamlines:(a)The Nuclear Microscopy (NM) beamline is mainly used for trace elemental quantitative mapping using a combination of Particle Induced X-ray Emission (PIXE), to measure the trace elemental concentration of inorganic elements, Rutherford Backscattering Spectrometry (RBS), to characterise the organic matrix, and Scanning Transmission Ion Microscopy (STIM) to provide information on the lateral areal density variations of the specimen. Typically, a 2.1MeV proton beam, focused to 1–2μm spot size with a current of 100pA is used.(b)The high resolution single cell imaging beamline is equipped with direct STIM to image the interior structure of single cells with proton and alpha particles of sub-50nm beam spot sizes. Simultaneously, forward scattering transmission ion microscopy (FSTIM) is utilized to generate images with improved contrast of nanoparticles with higher atomic numbers, such as gold nanoparticles, and fluorescent nanoparticles can be imaged using Proton Induced Fluorescence (PIF). Lastly, in this facility, RBS has been included as an option if required to determine the depth distribution of nanoparticles in cells, albeit with reduced spatial resolution.

Resist evaluation for proton beam writing, Ni mold fabrication and nano-replication

Proton beam writing (PBW) is a new direct-write technique which has shown great potential to fabricate structures down to 20 nm level in resist material. Protons can be accelerated up to a high energy (3.5 MeV) at Centre for Ion Beam Applications. Because the mass of a proton is much larger than the mass of an electron (mp:me = 1,800:1), the energy of the secondary electrons is very small compared with secondary electrons generated by electron beam lithography. Therefore, a proton will travel along a straight path into resist and secondary electrons will only expose the resist within several nanometers around the path of the proton. PBW is capable of fabricating structures with very straight, vertical and smooth sidewalls without proximity effect. This is very important when combining PBW with Ni electroplating and nanoimprinting as well as injection molding. High quality Ni molds with smooth and vertical side walls are critical in nanoimprint lithography and injection molding. In our experiments, several new resists including AR-P 3250, a mixture of AR-P 3250 and AR 300-12, and ma-N 2401 are tested with PBW for the production of high aspect ratio Ni molds and thermoplastic replication with these molds. High aspect ratio structures (up to 7) are fabricated at a width of 500 nm in Ni molds. The structures are transferred to plastic via nanoimprinting and injection molding.

Orthogonal and fine lithographic structures attained from the next generation proton beam writing facility

A second generation proton beam writing (PBW) system has been built at the Centre for Ion Beam Applications at the National University of Singapore for fabrication of high aspect ratio 3D nano lithographic structures. System improvements and a few lithographic structures obtained with this facility are presented in this paper. Through accurate alignment of the magnetic quadrupole lenses and the electrostatic scanning system, orthogonal beam scanning has been achieved. The earlier constrain of limited beam scan area has been overcome by adopting a combination of beam and stage scanning as well as stitching. With these improvements smallest ever Ni structure of 65 nm in width has been fabricated using nickel electroplating on a proton beam written PMMA sample in the second generation PBW facility. Using this improved PBW facility, we have also demonstrated the fabrication of fine lithographic patterns with 19 nm line width and 60 nm spacing in 100 nm thick negative high resolution hydrogen silsesquioxane resist. Future possible system improvements leading to finer resolution will be discussed briefly.

Sub‐100‐nm STIM imaging and PIXE quantification of rare earth elements in algae cells

The uptake of rare earth elements (REEs) such as Nd3+, Gd3+, and Er3+ by Euglena gracilis 848 algae cells has been studied using nuclear microprobe techniques at the Centre for Ion Beam Applications (CIBA), National University of Singapore. The cell imaging facility recently constructed at CIBA allows scanning transmission ion microscopy (STIM) down to the sub‐100‐nm level and provides energy loss images of whole intact cells. Helical coiling of the pellicle strips of the algae cells was observed, as was Rutherford backscattering spectrometry (RBS) images showing the distribution of erbium in the cell. Quantification of the REEs was carried out using a combination of nuclear techniques such as proton‐induced X‐ray emission, RBS, and STIM. Our results indicate significant variability in the uptake of REEs by algae cells. The amount of erbium absorbed was nearly one order of magnitude higher than that of neodymium, whereas the Gadolinium uptake was below the detection limit of 200 ppm. Copyright © 2013 John Wiley & Sons, Ltd.

An ion beam analysis software based on ImageJ

The suit of techniques (RBS, STIM, ERDS, PIXE, IL, IF,…) available in ion beam analysis yields a variety of rich information. Typically, after the initial challenge of acquiring data we are then faced with the task of having to extract relevant information or to present the data in a format with the greatest impact.This process sometimes requires developing new software tools. When faced with such situations the usual practice at the Centre for Ion Beam Applications (CIBA) in Singapore has been to use our computational expertise to develop ad hoc software tools as and when we need them. It then became apparent that the whole ion beam community can benefit from such tools; specifically from a common software toolset that can be developed and maintained by everyone with freedom to use and allowance to modify. In addition to the benefits of readymade tools and sharing the onus of development, this also opens up the possibility for collaborators to access and analyse ion beam data without having to depend on an ion beam lab. This has the virtue of making the ion beam techniques more accessible to a broader scientific community.We have identified ImageJ as an appropriate software base to develop such a common toolset. In addition to being in the public domain and been setup for collaborative tool development, ImageJ is accompanied by hundreds of modules (plugins) that allow great breadth in analysis. The present work is the first step towards integrating ion beam analysis into ImageJ. Some of the features of the current version of the ImageJ ‘ion beam’ plugin are: (1) reading list mode or event-by-event files, (2) energy gates/sorts, (3) sort stacks, (4) colour function, (5) real time map updating, (6) real time colour updating and (7) median & average map creation.

The Singapore high resolution single cell imaging facility

The Centre for Ion Beam Applications, National University of Singapore has recently expanded from three state-of-the-art beam lines to five. Two new beam lines have been constructed: A second generation proton beam writing line, and a high resolution single cell imaging facility. Both systems feature high demagnification lens systems based on compact Oxford Microbeams OM52 lenses, coupled with reduced lens/image distances.The single cell imaging facility is designed around OM52 compact lenses capable of operating in a variety of high demagnification configurations including the spaced Oxford triplet and the double crossover Russian quadruplet. The new facility has design specifications aimed at spatial resolutions below 50nm, with a variety of techniques including STIM, secondary electron and fluorescence imaging, and an in-built optical and fluorescence microscope for sample imaging, identification and positioning.Preliminary tests using the single space Oxford triplet configuration have indicated a beam spot size of 31×39nm in the horizontal and vertical directions respectively, at beam currents of ∼10,000 protons per second. However, a weakness in the specifications of the electrostatic scanning system has been identified, and a more stable scanning system needs to be implemented before we can fully realize the optimum performance. A single whole fibroblast cell has been scanned using 1.5MeV protons, and a median fit to the proton transmission energy loss data has shown that proton STIM gives excellent details of the cell structure despite the relatively poor contrast of proton STIM compared with alpha STIM.

Proton beam writing nanoprobe facility design and first test results

A second generation proton beam writing line has been installed at the Centre for Ion Beam Applications at the National University of Singapore. This system features a high demagnification lens system based on compact OM52 lenses with reduced working distance. The system can operate in a variety of high demagnification configurations, including the spaced Oxford triplet and the double-crossover Russian quadruplet. It features an electrostatic scanning system and a new collimator slit aperture design. Here we present the first results with this system operating in a spaced Oxford triplet configuration with a beam focus in one direction down to 20nm. One of the limitations of the current system is the inferior quality of the high-voltage supply driving the electrostatic scanning system. The second focus of this paper is the performance of the new collimator slit aperture design for MeV nanoprobe forming systems. Cylindrically strained electroplated Ni surfaces with a bending radius of 50mm are used as slit edges. An incident molecular beam of 2MeV H2+ ions was used for the slit scattering measurements. Any scattering by the slits resulting in dissociation of the incident molecular beam into 1MeV protons facilitated the measurements of scattered and non-scattered components by the passivated implanted planar silicon (PIPS) detector. The studies showed the expected correlation between the aperture size and scattering. The quality of the beam was satisfactory even for small aperture dimensions with a very small percentage of the beam showing energy loss.

A new data acquisition and imaging system for nuclear microscopy based on a Field Programmable Gate Array card

The introduction of the new Field Programmable Gate Array (FPGA) cards by National Instruments has made it possible for the first time to develop reconfigurable custom data acquisition hardware easily with the LabVIEW programming environment. Data acquisition issues such as precise timing for scanning and operating system latencies can now be easily overcome using this new technology because the data acquisition software is embedded in the FPGA chip on the card. In this paper we present the first results of the new data acquisition system developed at the Centre for Ion Beam Applications (CIBA), National University of Singapore using the new National Instruments cards in conjunction with rack mountable Wilkinson type ADCs.

Proton beam writing: a platform technology for nanowire production

P-beam writing (proton beam writing) is a unique direct write 3D nano-lithographic technique which has been developed at the Centre for Ion Beam Applications (CIBA), in the Physics Department of the National University of Singapore. This technique employs a focused MeV proton beam which is scanned in a predetermined pattern over a resist (e.g. PMMA, SU-8 or HSQ), which is subsequently chemically developed. In e-beam writing as well as p-beam writing the energy loss of the primary beam is dominated by energy transfer to substrate electrons. Unlike the high energy secondary electrons generated during e-beam writing the secondary electrons induced by the primary proton beam have low energies (typically less than 100 eV) and therefore a limited range, resulting in minimal proximity effects. The low proximity effects exhibited by p-beam writing coupled with the straight trajectory and high penetration of the proton beam enables the production of high aspect ratio, high density 3D micro- and nano-structures with well defined smooth side walls to be directly written into resist materials. These structures can be used as templates to electroplate metallic nanowires.

HRBS/channeling studies of ultra-thin ITO films on Si

High-resolution Rutherford backscattering spectroscopy (HRBS)/channeling techniques have been utilized for a detailed characterization of ultra-thin indium tin oxide (ITO) films and to probe the nature of the interface between the ITO film and the Si(001) substrate. Channeling studies provide a direct measure of the lattice strain distribution in the crystalline Si substrate in the case of amorphous over layers. The measurements on DC magnetron sputtered ITO films have been carried out using the recently installed HRBS facility at the Centre for Ion Beam Applications (CIBA). The thickness of the ultra-thin (∼9.8nm) ITO films was calculated from the HRBS spectra having an energy resolution of about 1.4keV at the superimposed leading (In+Sn) edge of the ITO film. The films were near stoichiometric and the interface between ITO film and Si was found to include a thin SiOx transition layer. The backscattering yields from (In+Sn) of ITO were equal in random and channeling directions, thereby revealing the non-crystalline nature of the film. Angular scans of HRBS spectra around the off-normal [111] axis clearly showed a shift in the channeling minimum indicative of compressive strain of the Si lattice at the SiOx/Si interface. The observed strain was about 0.8% near the interface and decreased to values below our detection limits at a depth of ∼3nm from the SiOx/Si interface.

Fabrication of nanofluidic devices utilizing proton beam writing and thermal bonding techniques

The fabrication of polymer lab-on-a-chip systems for applications in Chemistry and Biology is one of the envisaged niche areas for the Proton Beam Writing (PBW) technique developed at the Centre for Ion Beam Applications (CIBA). Utilizing a highly focused beam of MeV protons, well-defined nanostructures with smooth and straight side walls have been directly written in a 500 nm to 10 μm thick PMMA layer spin coated onto a Kapton substrate. By subsequently thermally bonding the fabricated structures to bulk PMMA and carefully peeling off the Kapton, nanostructures can be attached to bulk PMMA. Finally, by bonding a PMMA sheet to the bottom side of the structure, an integrated PMMA device with enclosed multiple high aspect ratio nanochannels can be realized. Preliminary experiments conducted in order to test this polymeric device indicate good fluidic properties. The nanochannels can be easily filled with dye solution using both pressure and capillary action in the case of hydrophilic solutions.

Nano-imaging of single cells using STIM

Scanning transmission ion microscopy (STIM) is a technique which utilizes the energy loss of high energy (MeV) ions passing through a sample to provide structural images. In this paper, we have successfully demonstrated STIM imaging of single cells at the nano-level using the high resolution capability of the proton beam writing facility at the Centre for Ion Beam Applications, National University of Singapore. MCF-7 breast cancer cells (American Type Culture Collection [ATCC]) were seeded on to silicon nitride windows, backed by a Hamamatsu pin diode acting as a particle detector. A reasonable contrast was obtained using 1 MeV protons and excellent contrast obtained using 1 MeV alpha particles. In a further experiment, nano-STIM was also demonstrated using cells seeded on to the pin diode directly, and high quality nano-STIM images showing the nucleus and multiple nucleoli were extracted before the detector was significantly damaged.

Nuclear microscopy measurement of copper in atherosclerosis – Sensitivity and limitations to spatial resolution

Nuclear microscopy studies at the Centre for Ion Beam Applications have indicated a link between iron, zinc and the development of atherosclerosis. In the present study, we have extended this study to copper, since copper is also capable of inducing free radical mediated damage. As copper in biological tissue occurs at the parts per million level and therefore close to the detection limits for PIXE analysis, we have substantially increased the beam intensity and scanning time to obtain adequate statistics and analytical sensitivity. The experiments were conducted on male New Zealand White rabbits, weighing approximately 2.5 kg and fed on high cholesterol diets for 8 weeks. Unlike iron concentrations, which were observed to be increased in the atherosclerotic lesion, our experiments show that copper is depleted in the lesion (1.9 ppm) compared with the adjacent artery wall (4.1 ppm). The concentration of copper present in the lesion is also much less than iron (approximately 30 times less on average), indicating that iron, rather than copper, is more likely to induce atherosclerosis through free radical mediated damage, purely on the basis of greatly reduced concentrations. To obtain adequate statistics for copper, a 2.1 MeV proton beam focused to 2 μm with a relatively high beam current (from about 800 pA to 1 nA) was scanned over the sample for approximately 4 h. The effects of beam damage due to these high beam currents have been investigated and it was found that the tissue shrinks 10% each in the X and Y direction contributing to total area shrinkage of 20%. Despite tissue shrinkage problems, our results indicate that even with 1 nA beam currents, 4 h scan times and scan sizes of around 1 × 1 mm 2, we can still extract accurate trace elemental concentrations.

The CIBA high resolution RBS facility

Recently, a high resolution RBS facility was installed at the Centre for Ion Beam Applications (CIBA) at the National University of Singapore (NUS). The magnetic spectrometer comprises of a 90° double focusing sector magnet and a 2D-MCP focal plane detector. The UHV scattering chamber is equipped with a five axis goniometer for channeling applications, the setup is similar to that used by the Kyoto group [K. Kimura, M. Kimura, Y. Mori, M. Maehara, H. Fukuyama, AIP Conf. Proc. 475 (1999) 500]. State of the art performance characteristics were expected, because the system operates in conjunction with the NUS Singletron accelerator, which provides ion beams with very favorable phase space characteristics. Well resolved spectra of 10nm SiO2/Si are shown, and an energy resolution of 0.9keV FWHM was measured at the leading Si edge of a SiO2 sample at 300keV and 80° scattering angle, which is close to the value expected from kinematic broadening alone in this case (∼0.7keV).

A progress review of proton beam writing applications in microphotonics

The proton beam writing technique is a direct write lithographic technique that is being actively developed at the Centre for Ion Beam Applications, National University of Singapore for microphotonic applications. The technique utilizes a highly focused beam of MeV protons to pattern or modify the refractive index of various materials including polymers, glasses and other inorganic crystals. The technique has been applied to the fabrication of several different types of microoptical components including waveguides, gratings, microlens arrays and colloidal crystal templates. In this paper we give a review of the progress made thus far by our group and other workers in the field of microphotonics using proton beam writing.

Ionscan: scanning and control software for proton beam writing

The proton beam writing technique relies on a precise beam scanning and control system that offers a simple yet flexible interface for the fabrication and design of microstructures. At the Centre for Ion Beam Applications, National University of Singapore, we have developed a suite of programs, collectively known as Ionscan, that cater for the specific needs of proton beam writing. The new version of Ionscan is developed using the Microsoft Visual C++. NET development environment in conjunction with a National Instruments analog output card and NI-DAQ drivers. With the benefit of the experience gained in proton beam writing over the years, numerous enhancements and new features have been added to the scanning software since the first version of the program that was developed using LabVIEW [A.A. Bettiol, J.A. van Khan, T.C. Sum, F. Watt, Nucl. Instr. and Meth. B 181 (2001) 49]. These include the ability to perform combined stage and magnetic (or electrostatic) scanning, which is particularly useful for the fabrication of long waveguides and microfluidic channels over lengths of up to 2.5cm. Other enhancements include the addition of the Ionutils program which gives the user the ability to design basic structures using an ASCII file format that was developed. This format contains basic information on the shape to be irradiated including the way in which it is scanned.

Proton beam writing: a progress review

A new direct write 3D nano lithographic technique has been developed at the Centre for Ion Beam Applications (CIBA) in the Physics Department of the National University of Singapore. This technique employs a focused MeV proton beam which is scanned in a predetermined pattern over a resist (e.g. PMMA or SU-8), which is subsequently chemically developed. The secondary electrons induced by the primary proton beam have low energy and therefore limited range, resulting in minimal proximity effects. Low proximity effects coupled with the straight trajectory and high penetration of the proton beam enables the production of 3D micro and nano structures with well-defined smooth side walls to be directly written into resist materials. In this review the current status of proton beam writing will be discussed; recent tests have shown this technique capable of writing high aspect ratio walls up to 160 and details down to 30 nm in width with sub-3 nm edge smoothness.