A gas ionisation Direct-STIM detector for MeV ion microscopy

Nuclear Instruments and Methods in Physics Research B 348 (2015) 58–61

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A gas ionisation Direct-STIM detector for MeV ion microscopy Rattanaporn Norarat a,b,c,⇑, Edouard Guibert a, Patrick Jeanneret a, Mario Dellea a, Josef Jenni a, Adrien Roux d, Luc Stoppini d, Harry J. Whitlow a a

University of Applied Sciences (HES-SO), Haute Ecole Arc Ingénierie, Eplatures-Gris 17, CH-2300 La Chaux-de-Fonds, Switzerland Faculty of Science and Agriculture, Rajamangala University of Technology Lanna, Chiang Rai, 57120 Chiang Rai, Thailand c Department of Physics, University of Jyväskylä, P.O. Box 35, Jyväskylä FI-40014, Finland d Tissue Engineering Laboratory, Campus Biotech, Chemin des Mines 9, Geneva, Switzerland b

a r t i c l e

i n f o

Article history: Received 21 July 2014 Received in revised form 20 December 2014 Accepted 24 December 2014 Available online 19 January 2015 Keywords: MeV ion microscope Direct-STIM Ionisation detector Bioimaging ReNcells VM

a b s t r a c t Direct-Scanning Transmission Ion Microscopy (Direct-STIM) is a powerful technique that yields structural information in sub-cellular whole cell imaging. Usually, a Si p-i-n diode is used in Direct-STIM measurements as a detector. In order to overcome the detrimental effects of radiation damage which appears as a broadening in the energy resolution, we have developed a gas ionisation detector for use with a focused ion beam. The design is based on the ETH Frisch grid-less off-axis Geiger–Müller geometry. It is developed for use in a MeV ion microscope with a standard Oxford Microbeams triplet lens and scanning system. The design has a large available solid angle for other detectors (e.g. proton induced fluorescence). Here we report the performance for imaging ReNcells VM with lm resolution where energy resolutions of <24 keV fwhm could be achieved for 1 MeV protons using isobutane gas. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction The practical limit of attainable resolution in ion microscopy is governed by the need to have a sufficiently large beam current and hence beam spot-size to generate a meaningful count rate in the detector. In Direct-Scanning Transmission Ion Microscopy (Direct-STIM), every ion traversing the sample is detected necessitating the use of sub-pfA beam currents. This allows the very small beam spot sizes to be realised through the use of small objective and collimator apertures, giving resolutions below 100 nm [1,2]. Further convincing evidence that these resolutions are achievable is that proton beam writing can produce structures down to 30 nm, or so, using low beam currents in quadrupole triplet lens configurations [3,4]. In conventional Direct-STIM imaging Si charged particle detectors are used for energy measurement. The focused ion beams in a MeV ion microscope implies that locally high ion fluences impinge on the detector surface giving rise to enhanced recombination [5] causing a progressive reduction in pulse height with increasing number of counts [1,6,7]. This manifests itself as an energy resolution degradation thereby limiting energy-loss image contrast.

⇑ Corresponding author at: Faculty of Science and Agriculture, Rajamangala University of Technology Lanna, Chiang Rai, 57120 Chiang Rai, Thailand. E-mail address: [email protected] (R. Norarat). http://dx.doi.org/10.1016/j.nimb.2014.12.074 0168-583X/Ó 2015 Elsevier B.V. All rights reserved.

Repeated use of Si charged particles detectors with different scan sizes and areas etc, will give rise to a spatially varying damage concentration that can even degrade lateral resolution [6]. Setting up the low current beam using a Si detector can easily lead to significant energy shifts and accidental direct exposure to nA beams inevitably destroys the Si detectors. Recently gas ionisation detectors have been developed for ion beam analytical applications [7,8]. These are resistant to radiation damage and can be recovered by replacing the gas. In this work we have investigated a compact high-pressure gas ionisation detector that is optimised for high spatial-resolution imaging with small solid angle and is not damaged by nA direct beams from the accelerator. 2. Detector configuration Fig. 1 shows the overall design of the detector. The entrance window was a 150 nm thick 1  1 mm Si3N4 membrane on a 5  5 mm Si frame 315 lm thick [9] which is mounted at the apex of the conical section. The detector is mounted on the 0 port of an Oxford Microbeams™ octagonal sample chamber so that the distance between the sample and entrance window is 5 mm. The sample-window distance should be as short as possible because of geometric considerations associated with the divergence of the beam behind the conjugate focus and beam scanning. The conical form of the end of the detector effectively allows a large unobscured solid angle about the target to facilitate operation with

R. Norarat et al. / Nuclear Instruments and Methods in Physics Research B 348 (2015) 58–61

Fig. 1. Schematic of the gas ionisation detector.

the off-axis STIM and proton induced fluorescence detectors. The vibration-free mounting of the target chamber and sealing-off the detector during operation, effectively suppressed microphonic pick-up from vacuum pumps etc. The detector has an off-axis Geiger–Müller geometry with a 1 mm dia. anode wire. This was operated with a high pressure (350–850 mbar) of isobutane as a filling gas to confine the ion–electron plasma produced by the ions to a small volume a cm in length and a few mm in lateral width for 0.5–2 MeV protons and 4He ions. This was verified using SRIM. 3. Electronics configuration Compared to Si charged particle detectors, the charge signal from gas ionisation detectors is about 10 times smaller. Measurement of 1 MeV protons with 1% fwhm electronic width corresponds to an electronic noise contribution (ENC) at the input of only 125 e-rms. To minimise noise and pick-up, meticulous care was needed. In particular, capacitive coupling of high frequency noise had to be eliminated. To minimise pick-up from ground loops, the body of the detector (cathode) was insulated from the sample chamber and the cathode connected to ground only through the pre-amplifier–main-amplifier–data acquisition chain. Also it was found the anode wire had to be completely shielded by the cathode from the target chamber. The charge-sensitive preamplifier used was an Ortec 142IH that was coupled to a Tennelec TC244 spectroscopy amplifier. This was connected to a standard Oxford Microbeams™ data acquisition system. The bias voltage was supplied by a Canberra 3106D high voltage supply. A pulser (tail-pulse generator) was connected in series with a shielded ceramic calibration capacitor to absolutely calibrate the detector system by applying pulses of precisely known charge to the pre-amplifier input. The pulser voltage step amplitudes were measured using a Tektronix TDS 220 oscilloscope by averaging over 64 pulses. In this way the pulse height in channels could be calibrated in terms of the absolute number of collected electrons. The calibration capacitor was calibrated to be 1.03  0.01 pF. Connector stray impedances make such small capacitors difficult to measure using an rf bridge method. Therefore the capacitor was calibrated over a range of kHz frequencies by using the fundamental relation, iðtÞ ¼ CdV=dt. The square wave rms current irms through the capacitor was measured using a 5 kX load resistance in series with the capacitor when a sawtooth voltage with constant j dV=dt j and periodic alternating sign, was applied across the CR circuit. A lock-in amplifier was used to measure the extremely small (lV) voltage developed across the load resistance.

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average charge carrier drift velocity to the mean time between collisions with a gas molecule. This was measured with a main amplifier peaking time of 4 ls and triangular shaping. Then j is then a measure of the probability of electron capture in the case that the field is too low for multiplication. The charge collection efficiency has been normalised to the saturation value which was taken to be when the pulse height varies by less than 0.5% over a 50 V bias voltage step. The charge collection in Fig. 2(a) shows that for all pressures the charge collection approached a constant asymptotic value as the bias voltage increased. Furthermore, it can be seen from Fig. 2(a) that the data points for 750 and 850 mbar are closely clustered about the same line. This is not the case for lower pressures. Using simple field models based on coaxial cylinder geometry with offaxis ion track and ion mobility data from Table 4.2 of Sauli’s compilation [11], estimated collection times are a few hundred nanoseconds and 1 ms for electrons and ions, respectively. Even after increasing these by factors of 5 to include the increases in these values associated with the lower electric field in the conical window region, the electron collection time is much shorter than the main-amplifier peaking time, while the ion collection time is much longer. Then with a wide margin the pulse height is mainly contributed to by the collected electrons. (<0.3% contribution from ions). Then under these conditions a plateau in collected charge with j as seen in Fig. 2 corresponds to complete charge collection in the asymptote. At low pressures the asymptotic value in Fig. 2 is reached at higher j. As the pressure is increased, the asymptotic charge collection value is reached at lower j. This behaviour is opposite to what is expected if charge recombination dominates the response. The ion range extends longer into the detector as the pressure is

4. Results and discussions 1 MeV protons were used for testing the performance of the gas ionisation detector. These have the same stopping force as 2 MeV Hþ 2 which was used previously [1,2,10]. Fig. 2(a) shows the charge collection efficiency dependence on j, the ratio of bias voltage to pressure. j is related to the ratio of

Fig. 2. (a) Charge collection and (b) resolution vs j for different isobutane pressures. The charge is normalised as described in the text and the peaking time of the main amplifier was 4 ls. In (a) the error bars are the same size as the data points. The scanned area is 30  30 lm.

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R. Norarat et al. / Nuclear Instruments and Methods in Physics Research B 348 (2015) 58–61

Fig. 3. Mean energy loss Direct-STIM image of ReNcells. The image has been subjected to a 2  2 median filter and the colourmap selected to optimise contrast. Increasing mean energy loss corresponds to the sequence: orange–yellow–green– blue-black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

decreased, (44.3 mm at 350 mbar decreasing to 18.3 mm at 850 mbar). If the charge collection efficiency is lower for short range ions because of the geometry in proximity to the entrance window, one may expect the asymptotic value is reached at higher bias voltages. This contrary to what is observed in the plots of charge collection with bias voltages (data not shown). In terms of j, the pressure scaling of range implies that the data in Fig. 2(a) should show the asymptotic value is reached at higher j, which again, is contrary to what is observed. This infers, that despite the sub-optimal geometry, the longitudinal component of the electric field in the vicinity of the window achieves an efficient electron collection. The absolute value of the asymptotic charge collection increased slightly with increasing pressure (data not shown). This again is contrary to what is expected if the geometry has poor charge collection close to the entrance window. The clustering of the data points in Fig. 2(a) along the same line indicates this asymptote represents the 100% charge collection efficiency. After applying a negligible correction for energy loss in the window and non-ionising nuclear energy deposition processes this

corresponded to an average ion–electron pair formation energy in our detector, W of 26.04  0.46 eV for 1 MeV protons in isobutane. This is in close agreement with the tabulated evaluated values of 26.7 eV [12] for 3.8 MeV 4He ions with similar velocity to 1 MeV protons. However, measured values of W may depend on energy and ion species [12]. The origin of the small increase in absolute asymptotic charge collection is unclear. It might originate from charging of the Si3N4 membrane and the insulators needed to support the anode wire. Under the high isobutane pressure used in the detector the absence, of a Frisch grid will not give a position dependence of pulse height at the main amplifier output because the pulse height is almost completely due to electron collection. Moreover, the application for a microbeam implies the impinging ions represent a point source of ions at the entrance window so the ionisation distributions are largely invariant. Fig. 2(b) shows the j dependence of the resolution characterised in terms of the fwhm. The fitted straight lines are to guide the eye. It is clear from Fig. 2(b), that there is an improvement in resolution as the pressure is increased. However, no statistically significant dependence on j, and hence bias voltage is observed, for all pressures. The resolution dependence on main amplifier shaping was investigated and it was found that triangular shaping with 4 ls peaking time was optimum. For shorter peaking times, the resolution degraded due to the shot noise ENC, while for longer peaking times than 4 ls, the resolution was constant. One might expect if spatial variations in the ionisation distributions from one ion to another influenced the shape of the collected pulse, this would give rise to a shaping-time constant dependent resolution which was not observed. Using the pulser the ENC contribution corresponded to 17.8 keV fwhm. Bohr energy straggling in the entrance window, which should be a reliable estimate for 1 MeV protons contributes 0.108 keV fwhm, which can be neglected. Subtracting these contributions in quadrature from the detector fwhm yields a residual detector contribution of 16 keV fwhm. Hence the dominating contribution to the energy width of the signal comes from the ENC. In practical high resolution work where the scanned area is small; even a small number of ions give significant impingement fluxes in the detector. Reference to Fig. 6 in [7] shows that a 30 keV shift in peak position for a Si detector is reached after about 2  1013 400 keV protons cm2. This corresponds to only one image with 2  107 protons for a 10  10 lm scan size and a detector in close proximity to the sample. The situation is even worse for 4He ions [1]. Hence, for high resolution work, even the present 23 keV fwhm resolution with stable response represents a considerable improvement in effective resolution over that obtained with Si p-i-n diodes.

Table 1 Protocol for preparation of ReNcells sample. Basic Medium: was prepared from DMEM/F-12 with GlutaMAX medium [14] and B27 neural cell supplement mix [15], Penicilline/Streptomycin [16], and Heparin [17] Differentiation of neural stem-cells: ReNcells VM Human Neural Progenitor Cell Line [18] cells were switched from proliferating medium (Basic medium supplemented with bFGF [19] and EFG [20]) to differentiating medium (Basic Media supplemented with cAMP [21]). Cells were differentiated in culture for at least 14 days (37 °C humidified air with 5% CO2) Seeding and culture: Differentiated ReNcells VM were detached by trypsinising in an incubator at 37 °C and fetal bovine serum was added to inhibit the action of the enzyme. The cells were centrifuged for 3 min at 1200 rpm and the pellet of cells was resuspended in differentiating media. The cells concentration was adjusted to 5.4  104 cells ml1 in a differentiating media containing 5 lg ml1 laminin. The Si3N4 membranes on a Si frames were sterilised by introducing them vertically and immersing them 70% ethanol for 5 min followed by rinsing (2) is sterile water. 50 ll of the cells suspension were seeded on the centre of the Si3N4 membrane in a culture well and incubated for 2 h at 37 °C in humidified air with 5% CO2. After the adhesion of the cells, medium was added in order to fully recover the membrane and were incubated for 14 days before imaging. Cells were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature Drying method: Following [2], the membrane with attached cells were immersed in 50:50 v/v methanol:water for 2 min (2), 100% methanol for 2 min (3), and then twice in 50:50 methanol:tetrabutanol for 2 min, followed with 100% tetrabutanol for 10 min then in hexamethyldisilazane for 3 min followed by drying in filtered air.

R. Norarat et al. / Nuclear Instruments and Methods in Physics Research B 348 (2015) 58–61

An image of the mean energy loss in ReNcells VM [13] is presented in Fig. 3. The 1 MeV beam of 400 protons s1 was focused to a beam spot size of 0.8  1.84 lm, determined using a Cu TEM grid-bar edge. The fluence was 1:2  1011 ions cm2. The cells were cultured and air-dried on 100 nm Si3N4 membranes according to the protocol in Table 1. The absence of a damage-induced energy shift allows even small changes in the mean energy loss to be determined even though the energy resolution for this measurement was poor (31 keV fwhm). This can be seen in the circa 3 lm wide axion extending between the two cell clusters. The absence of a change in background colour over the image indicates that the charge collection is independent of position even though no Frisch grid is used. 5. Conclusions A compact high-pressure gas ionisation detector for high-resolution Direct-STIM imaging has been developed. The detector overcomes the problems associated with ion induced changes in the detector response for conventional Si detectors. For 1 MeV protons a saturation in the charge collection at a pressure of P750 mbar occurred for anode voltages of 600 V and above. This corresponded to a measured mean energy deposition to create an ion–electron pair in pure isobutane of 26.0  0.4 eV. The optimal resolution of the detector for 1 MeV protons was 24  1.7 keV fwhm. It can be improved by using electronics with a lower ENC. The detector has been successfully used for imaging neural cells grown from ReN cells VM. Acknowledgements The work has been supported by HES-SO project: 34940. R.N. is grateful for support from the Magnus Erhnrooth Foundation, the Academy of Finland Center of Excellence in Nuclear and Accelerator Based Physics (Ref. 251353) and Rajamangala University of Technology Lanna. References [1] Ren Minqin, J.A. van Kan, A.A. Bettiol, Lim Daina, Chan Yee Gek, Bay Boon Huat, H.J. Whitlow, T. Osipowicz, F. Watt, Nano-imaging of single cells using STIM, Nucl. Instr. Meth. B 260 (2007) 124.

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[2] R. Norarat, V. Marjomäki, X. Chen, M. Zhaohong, R. Minqin, C. Chen, A.A. Bettiol, H.J. Whitlow, F. Watt, Ion-induced fluorescence imaging of endosomes, Nucl. Instr. Meth. B 306 (2013) 113. [3] J.A. van Kan, A.A. Bettiol, F. Watt, Three-dimensional nano lithography using proton beam writing, Appl. Phys. Lett. 83 (2003) 1629. [4] A.A. Bettiol, J.A. van Kan, E.J. Teo, M.B.H. Breese, Ion beam lithography and nanofabrication : a review, Int. J. Nanosci. 4 (2005) 269. [5] Y. Zhang, T. Winzell, H.J. Whitlow, Influence of heavy ion irradiation damage on Si charged particle detector calibration, Nucl. Instr. Meth. B 161–163 (2000) 297. [6] D.P.L. Simons, A.J.H. Maas, P.H.A. Mutsaers, M.J.A. de Voigt, Study of localised radiation damage to PIPS detectors by a scanning ion microprobe: measured effects and consequences for STIM analysis, Nucl. Instr. Meth. B 130 (1997) 160–165. [7] A.M. Müller, A. Cassimi, M. Döbeli, M. Mallepell, I. Monnet, M.J. Simon, M. Suter, H.-A. Synal, A new mini gas ionization chamber for IBA applications, Nucl. Instr. Meth. B 269 (2011) 30–37. [8] A.C. Marques, M.M.F.R. Fraga, P. Fonte, D.G. Beasley, L.C. Alves, R.C. da Silva, New gas detector setup for on-axis STIM tomography experiments, Nucl. Instr. Meth. B 306 (2013) 104–108. [9] Silson Ltd., Northampton, UK, . [10] H.J. Whitlow, M. Ren, J.A. van Kan, F. Watt, D. White, Exploratory nuclear microprobe data visualisation using 3- and 4-dimensional biological volume rendering tools, Nucl. Instr. Meth. B 260 (2007) 28. [11] F. Sauli, Gaseous radiation detectors: Fundamentals and applications, in: Cambridge Monographs on Particle Physics, Nuclear Physics and Cosmology, 36, Cambridge University Press, 2014. [12] Average energy required to produce an ion pair, International Commission on Radiation Units and Measurements, ICRU Report No. 31, Washington, DC, 1979. [13] R. Donato, E.A. Miljan, S.J. Hines, S. Aouabdi, K. Pollock, S. Patel, F.A. Edwards, J.D. Sinden, Differential development of neuronal physiological responsiveness in two human neural stem cell lines, BMC Neurosci. 8 (2007) 36. [14] . [15] B27 neural cell supplement mix, Life Technologies, . [16] PAA . [17] Heparin, Sigma, . [18] Merck Millipore, # SCC008, . [19] bFGF Peprotech, . [20] EFG Peprotech, . [21] cAMP, Sigma, .