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Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy
Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy Sudheer Kumar Vanga ⇑, Zhaohong Mi, Long Cheng Koh, Ye Tao, Andrew A. Bettiol, Frank Watt Centre for Ion Beam Applications, Department of Physics, National University of Singapore, 2 Science Drive 3, Singapore 117551, Singapore
a r t i c l e
i n f o
Article history: Received 25 July 2014 Received in revised form 28 January 2015 Accepted 30 January 2015 Available online xxxx Keywords: Ion beam induced fluorescence Scanning transmission ion microscopy Parabolic mirror Fluorescence detection
a b s t r a c t Ion beam induced fluorescence microscopy is a new imaging technique which has the potential to achieve sub-50 nm spatial resolution fluorescence images. Currently the resolution of the technique has been limited to around 150 nm mainly because of inefficient collection and detection of emitted photons from the sample. To overcome this limitation, a new light collection system based on a custom made parabolic mirror is employed to enhance the fluorescence collection. The custom made mirror is designed so as to obtain both structural (scanning transmission ion microscopy) and ion beam induced fluorescence imaging simultaneously. The design and characterization of the parabolic mirror is discussed in detail. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Ion beam induced fluorescence together with ion beam characterization techniques such as particle induced X-ray emission (PIXE) and Rutherford backscattering spectroscopy (RBS) has been used to determine defects in crystal structure in solid materials and to study the chemical bonds in organic materials [1–3]. More recently the technique has been applied for bio-imaging such as to observe the autofluorescence from cryosectioned skin tissues [4] and to study the non-fluorescent and fluorescent tissue stains in common pathological tissue [5]. In our previous studies, using a focused MeV proton beam, fluorescence images of N2A blastoma cells stained with Sytox green and A549 lung carcinoma cells, stained with EGFR and receptor a2b1 integrin, was obtained at the single cell level with resolutions 200 nm or better [6,7]. Although the state-of-the-art spatial resolutions achieved are of the order of 20 nm using MeV ions [8], the same spatial resolutions have not been achieved in ion beam induced fluorescence microscopy. Though the technique has been applied to single cell imaging, the ion beam induced fluorescence resolution is limited to 150 nm whereas in scanning transmission ion microscopy (STIM) spatial resolution of 25 nm is reported [9]. There are two main reasons for the difference in spatial resolutions between STIM imaging and ion beam induced fluorescence imaging, one is the lack of efficient radiation stable fluorescent probes and the other is poor light collection system. Because of these limitations, ⇑ Corresponding author. E-mail address: [email protected] (S.K. Vanga).
the imaging requires high ion beam currents for the fluorescence detection resulting in higher spatial resolution compared to STIM. The current system in our cell imaging facility at Centre for Ion Beam Applications at the National University of Singapore, utilizes a light collection detector placed behind the cell sample which allows the detector to collect only a portion of emitted light (forward scattered light). Overcoming this light collection limitation will allow for improved resolution fluorescence imaging well below the 100 nm level. Furthermore, combining ion beam induced fluorescence imaging with structural imaging using STIM will provide potentially new information which will improve our understanding of cell function at the sub-cellular level. In this paper, we focus on improving the ion beam induced fluorescence light collection system with a custom made parabolic mirror. 2. Parabolic mirror design A parabolic reflector is able to reflect light emitted from an isotropic point source positioned at its focal point into parallel rays traveling in the direction of the parabolic axis. These parallel rays of emitted light from the sample can be focused on to a detector which enables the collection of isotropic emission from the sample. However, a complete parabolic mirror can not be implemented in the set-up since the ion beam has to travel through the mirror and interact with the sample to emit the fluorescence. One of the major challenges is accommodating the parabolic mirror in the current system. The specific constraints in designing the parabolic mirror are listed here.
http://dx.doi.org/10.1016/j.nimb.2015.01.076 0168-583X/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
2
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
Fig. 1. Parabolic mirror design parameters (a) Final custom-made mirror showing the individual parts. (b) Schematic showing the design parameters and (c) comparison of the fluorescence collection efficiency of the existing set-up and custom-made parabolic mirror with non-reflective and reflective sample and target holder obtained from ray tracing simulations.
Fig. 2. Ion beam induced fluorescence microscope set-up. (a) Schematic of the set-up; (b) shows the experimental set-up indicating the individual components (c) shows the magnified view of the rectangular area indicated in (b).
For high demagnification geometries, the target needs to be close to the lens and hence near to the target chamber wall. This sets a limit on the size of the parabolic mirror. The target needs to be positioned at the focal point of the mirror system, and this requires a suitable opening in the mirror.
Since the focal point of the parabolic mirror lies in the principle axis, the position of the opening should be asymmetric. For STIM imaging, the focused ion beam passes through a 2 mm hole in the front of the mirror, transmits through the sample, and pass through a similar hole in the back portion of the mirror.
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
3
Fig. 3. Parabolic mirror characterization results showing (a) the depth of focus of the mirror and (b) the field of view of the mirror in X and Y directions, color scale indicates the number of photons collected. (c) off-axis STIM and (d) ion beam induced fluorescence images of the lanthanide doped nanorods obtained simultaneously after incorporating the parabolic mirror in the set-up. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The parabolic mirror is designed by considering the above mentioned constraints. The complete mirror is designed as three separate parts for flexible operation, the major mirror, minor mirror and mirror holder. The mirror hole is designed such that the focal point of the parabolic mirror lies in the center of the 2 mm mirror hole in both X and Y directions. The asymmetric rectangular opening is designed such that the focal point in Z direction is 1 mm away from the major mirror. The mirror holder is designed such that the two detachable mirror parts can be attached to one end and the standard 1-inch SM1 (Thorlabs) tube can be attached to other end. With the SM1 tube mounting, additional optical components can be incorporated for further operations such as focusing the collimated light from mirror onto the detector, filtering light output, or collecting light into a fiber to guide the light out of the vacuum chamber. The mirror is fabricated in non magnetic 6061 aluminum alloy, which is both light-weight and durable. The inner portion of the mirror is diamond turned for high reflection. The drawing and the fabricated mirror can be seen in Fig. 1(a) and (b). Due to the necessary introduction of a rectangular cut and holes in the mirror, the mirror cannot act as a perfect reflector. Ray tracing simulations were performed to identify the modified parabolic mirror performance. The 3 dimensional design of the resulted mirror was modeled using Solid works and the design file was exported into Zemax simulation package [10] to perform ray tracing simulations. A point source was positioned at the focal point of the mirror and the detector was placed at the exit of the mirror to measure the reflected photons from the mirror. Fig. 1(c) shows the simulation results obtained for two scenarios; one assuming Table 1 A comparison of ion beam induced fluorescence counts obtained using different collection systems. Collection system
Fluorescence counts
Ratio
PMT with parabolic mirror PMT
37,979 16,754
2.3
the target holder and sample as non-reflective and the other as reflective. The results are also compared with the existing set-up in which no mirror was present and the detector was placed behind the sample. The maximum and minimum light collection efficiency of parabolic mirror system was projected to be 63% and 24% respectively which is higher than simulated efficiency of the existing set up (17%).
3. Experimental results The existing light collection system was modified by introducing the custom-made parabolic mirror in the set-up. The parabolic mirror was aligned perpendicular to the beam axis and was placed on precision closed loop three axis stage (Mechonics Model MS-030) to move the mirror such that the focal point of the parabolic mirror coincides with the beam focus on the sample. The closed loop stage is controlled by the stage controller: CU30CL. The light-weight closed loop stage was attached to the main stage (open loop three axis stage, PI N-310K059) to which the target holder was mounted. The schematic and the actual set-up is shown in Fig. 2. The closed loop stage has 25 mm translation in all three axis with position accuracy of 50 nm and is chosen as light weight in order to reduce the load on the main stage. The mirror characterization experiments were carried out with a focused 1.6 MeV helium ion beam. The ion beam induced fluorescence was obtained by scanning the focused beam onto the lanthanide doped nano rods (NaYF4: 2% Tm 60% Yb) which were mounted on a 100 nm thick silicon nitride window. These upconversion nanoparticles produce blue and red emission (peaks at 450, 470 and 650 nm) under the 980 nm laser excitation. In this experiment the isotropic emission was collected using the parabolic mirror. The collimated fluorescence light from the parabolic mirror was focused onto a 600 lm diameter fiber using a 2 inch focal length plano-convex lens. The emitted light was guided out of the vacuum chamber through the fiber and was
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
4
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
Fig. 4. The ion beam induced fluorescence images of lanthanide doped nanorods collected using (a) the custom-made parabolic mirror together with a focusing mirror and PMT and (b) with a PMT placed behind the sample.
connected to the photomultiplier tube (PMT) (Hamamatsu PMT R7401P) for the detection of fluorescence emission. For the characterization of the fabricated custom-made mirror, first the mirror was translated such that the focal point of the parabolic mirror approximately coincides the ion beam focus on the sample. The ion beam was scanned electrostatically in an area of 54 lm by 54 lm and the mirror stage was scanned in Z direction by observing the fluorescence counts from the PMT. A plot of the fluorescence counts as a function of mirror position in Z direction can be seen in Fig. 3(a). The full width at half maximum (FWHM) of the plot gives the depth of the focus (DoF) of mirror which is 80 lm. Once the focal point in Z direction was determined, the stage was moved to the optimal Z position and raster scanned in X and Y directions to find the field of view (FoV) of the mirror. Fig. 3(b) shows the intensity plot of XY scan as a function of fluorescence counts. The FWHM of these plots gives the field of view of the mirror in X and Y directions which is 96 lm and 110 lm respectively. The cell imaging work at our facility requires a scan area of 50 lm by 50 lm which is within the mirror field of view. The simultaneous ion beam induced fluorescence imaging and scanning transmission ion microscopy imaging capability of parabolic mirror was tested by imaging the same lanthanide doped fluorescent nano rods. The STIM detector was translated and positioned behind the mirror hole and ion beam was scanned in an area of 13.2 lm by 13.2 lm. Fig. 3(c) and (d) shows the images of the off-axis STIM and ion beam induced fluorescence obtained simultaneously. To detect sufficient number of fluorescence counts using the system, ion beam charge collection was adjusted so that 165 alpha particles incident on the sample for each pixel in the image. The comparison between the existing collection system and the new parabolic mirror collection system was performed by obtaining the fluorescence images of the same lanthanide doped nanorods. The PMT used in both cases is the same (Hamamatsu PMT R7401P), which has a detection wavelength range is 300– 650 nm. The fluorescence counts was normalized with the STIM counts and the results were tabulated in Table 1. The collected images can be seen in Fig. 4. It is clear that the introduction of parabolic mirror enhances the light collection by a factor of 2.3, though the ray tracing simulation showed a factor of 3.7 improvement. This difference is reasonable since the target holder and the sample is not 100% reflective. Over the time aluminum may oxidize hence reduce the reflectivity so we plan to coat the mirror inner surfaces with a reflective coatings and also a protective coating to avoid oxidation. Future work will be focused on the comparison of ion beam induced fluorescence emission from different fluorescent probes and their radiation stability.
4. Conclusion In conclusion, a custom made parabolic mirror has been designed to improve the collection efficiency of ion beam fluorescence imaging. The custom-made mirror is installed in the current set-up and characterized experimentally to obtain the mirror characteristics such as depth of focus and field of view of the mirror. When compared to the existing collection system, the collection efficiency was improved by a factor of 2.3 by introducing the new parabolic mirror in the system. Adding this component to the set-up not only enhances the fluorescence collection but also allows us to obtain the STIM images simultaneously. Acknowledgement This work is supported by the Ministry of Education - Singapore, Academic Research Fund Tier 2 grant (R-144-000-306-112). The authors would like to acknowledge Xiaogang Liu and Yuhai Zhang from the Department of Chemistry for supplying the lanthanide doped nanorods. References [1] K.G. Malmqvist, M. Elfman, G. Remond, C. Yang, PIXE and ionoluminescence – a synergetic analytical combination, Nucl. Instr. Meth. B 109–110 (1996) 227– 233. [2] J. Zuk, T.J. Ochalski, M. Kulik, J. Lis, A.P. Kobzev, Effect of oxygen implantation on ionoluminescence of porous silicon, J. Lumin. 80 (1999) 187–192. [3] A.A. Bettiol, D.N. Jamieson, S. Prawer, M.G. Allen, Ion beam induced luminescence from a nuclear microbeam from diamond and other crystals, Nucl. Instr. Meth. B 85 (1994) 775–779. [4] J. Pallon, C. Yang, R.J. Utui, M. Elfman, K.G. Malmqvist, P. Kristiansson, K.A. Sjoland, Ionoluminescence technique for nuclear microprobes, Nucl. Instr. Meth. B 130 (1–4) (1997) 199–203. [5] P. Rossi, C.D. Maggio, G.P. Egeni, A. Galligioni, G. Gennaro, L. Giacomelli, A. Lo Giudice, M. Pegoraro, L. Pescarini, V. Rudello, E. Vittone, Cytological and histological structures identification with the technique IBIL in elemental microanalysis, Nucl. Instr. Meth. B 181 (2001) 437–442. [6] F. Watt, A.A. Bettiol, J.A. van Kan, M.D. Ynsa, R. Minqin, R. Rajendran, C. Huifang, S. Fwu-Shen, A.M. Jenner, Imaging of single cells and tissue using MeV ions, Nucl. Instr. Meth. B 267 (12–13) (2009) 2113–2116. [7] R. Norarat, V. Marjomäki, X. Chen, M. Zhaohong, R. Minqin, C.-B. Chen, A.A. Bettiol, H.J. Whitlow, F. Watt, Ion-induced fluorescence imaging of endosomes, Nucl. Instr. Meth. B 306 (2013) 113–116. [8] J.A. van Kan, P. Malar, A.B. de Vera, The second generation Singapore high resolution proton beam writing facility, Rev. Sci. Instrum. 83 (2) (2012) 02B902. [9] F. Watt, X. Chen, C.-B. Chen, C.N.B. Udalagama, J.A. van Kan, A.A. Bettiol, Whole cell structural imaging at 20 nanometre resolutions using MeV ions, Nucl. Instr. Meth. B 306 (2013) 6–11. [10] ZEMAX Optical Design Program: User’s Guide,
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy Sudheer Kumar Vanga ⇑, Zhaohong Mi, Long Cheng Koh, Ye Tao, Andrew A. Bettiol, Frank Watt Centre for Ion Beam Applications, Department of Physics, National University of Singapore, 2 Science Drive 3, Singapore 117551, Singapore
a r t i c l e
i n f o
Article history: Received 25 July 2014 Received in revised form 28 January 2015 Accepted 30 January 2015 Available online xxxx Keywords: Ion beam induced fluorescence Scanning transmission ion microscopy Parabolic mirror Fluorescence detection
a b s t r a c t Ion beam induced fluorescence microscopy is a new imaging technique which has the potential to achieve sub-50 nm spatial resolution fluorescence images. Currently the resolution of the technique has been limited to around 150 nm mainly because of inefficient collection and detection of emitted photons from the sample. To overcome this limitation, a new light collection system based on a custom made parabolic mirror is employed to enhance the fluorescence collection. The custom made mirror is designed so as to obtain both structural (scanning transmission ion microscopy) and ion beam induced fluorescence imaging simultaneously. The design and characterization of the parabolic mirror is discussed in detail. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Ion beam induced fluorescence together with ion beam characterization techniques such as particle induced X-ray emission (PIXE) and Rutherford backscattering spectroscopy (RBS) has been used to determine defects in crystal structure in solid materials and to study the chemical bonds in organic materials [1–3]. More recently the technique has been applied for bio-imaging such as to observe the autofluorescence from cryosectioned skin tissues [4] and to study the non-fluorescent and fluorescent tissue stains in common pathological tissue [5]. In our previous studies, using a focused MeV proton beam, fluorescence images of N2A blastoma cells stained with Sytox green and A549 lung carcinoma cells, stained with EGFR and receptor a2b1 integrin, was obtained at the single cell level with resolutions 200 nm or better [6,7]. Although the state-of-the-art spatial resolutions achieved are of the order of 20 nm using MeV ions [8], the same spatial resolutions have not been achieved in ion beam induced fluorescence microscopy. Though the technique has been applied to single cell imaging, the ion beam induced fluorescence resolution is limited to 150 nm whereas in scanning transmission ion microscopy (STIM) spatial resolution of 25 nm is reported [9]. There are two main reasons for the difference in spatial resolutions between STIM imaging and ion beam induced fluorescence imaging, one is the lack of efficient radiation stable fluorescent probes and the other is poor light collection system. Because of these limitations, ⇑ Corresponding author. E-mail address: [email protected] (S.K. Vanga).
the imaging requires high ion beam currents for the fluorescence detection resulting in higher spatial resolution compared to STIM. The current system in our cell imaging facility at Centre for Ion Beam Applications at the National University of Singapore, utilizes a light collection detector placed behind the cell sample which allows the detector to collect only a portion of emitted light (forward scattered light). Overcoming this light collection limitation will allow for improved resolution fluorescence imaging well below the 100 nm level. Furthermore, combining ion beam induced fluorescence imaging with structural imaging using STIM will provide potentially new information which will improve our understanding of cell function at the sub-cellular level. In this paper, we focus on improving the ion beam induced fluorescence light collection system with a custom made parabolic mirror. 2. Parabolic mirror design A parabolic reflector is able to reflect light emitted from an isotropic point source positioned at its focal point into parallel rays traveling in the direction of the parabolic axis. These parallel rays of emitted light from the sample can be focused on to a detector which enables the collection of isotropic emission from the sample. However, a complete parabolic mirror can not be implemented in the set-up since the ion beam has to travel through the mirror and interact with the sample to emit the fluorescence. One of the major challenges is accommodating the parabolic mirror in the current system. The specific constraints in designing the parabolic mirror are listed here.
http://dx.doi.org/10.1016/j.nimb.2015.01.076 0168-583X/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
2
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
Fig. 1. Parabolic mirror design parameters (a) Final custom-made mirror showing the individual parts. (b) Schematic showing the design parameters and (c) comparison of the fluorescence collection efficiency of the existing set-up and custom-made parabolic mirror with non-reflective and reflective sample and target holder obtained from ray tracing simulations.
Fig. 2. Ion beam induced fluorescence microscope set-up. (a) Schematic of the set-up; (b) shows the experimental set-up indicating the individual components (c) shows the magnified view of the rectangular area indicated in (b).
For high demagnification geometries, the target needs to be close to the lens and hence near to the target chamber wall. This sets a limit on the size of the parabolic mirror. The target needs to be positioned at the focal point of the mirror system, and this requires a suitable opening in the mirror.
Since the focal point of the parabolic mirror lies in the principle axis, the position of the opening should be asymmetric. For STIM imaging, the focused ion beam passes through a 2 mm hole in the front of the mirror, transmits through the sample, and pass through a similar hole in the back portion of the mirror.
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
3
Fig. 3. Parabolic mirror characterization results showing (a) the depth of focus of the mirror and (b) the field of view of the mirror in X and Y directions, color scale indicates the number of photons collected. (c) off-axis STIM and (d) ion beam induced fluorescence images of the lanthanide doped nanorods obtained simultaneously after incorporating the parabolic mirror in the set-up. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The parabolic mirror is designed by considering the above mentioned constraints. The complete mirror is designed as three separate parts for flexible operation, the major mirror, minor mirror and mirror holder. The mirror hole is designed such that the focal point of the parabolic mirror lies in the center of the 2 mm mirror hole in both X and Y directions. The asymmetric rectangular opening is designed such that the focal point in Z direction is 1 mm away from the major mirror. The mirror holder is designed such that the two detachable mirror parts can be attached to one end and the standard 1-inch SM1 (Thorlabs) tube can be attached to other end. With the SM1 tube mounting, additional optical components can be incorporated for further operations such as focusing the collimated light from mirror onto the detector, filtering light output, or collecting light into a fiber to guide the light out of the vacuum chamber. The mirror is fabricated in non magnetic 6061 aluminum alloy, which is both light-weight and durable. The inner portion of the mirror is diamond turned for high reflection. The drawing and the fabricated mirror can be seen in Fig. 1(a) and (b). Due to the necessary introduction of a rectangular cut and holes in the mirror, the mirror cannot act as a perfect reflector. Ray tracing simulations were performed to identify the modified parabolic mirror performance. The 3 dimensional design of the resulted mirror was modeled using Solid works and the design file was exported into Zemax simulation package [10] to perform ray tracing simulations. A point source was positioned at the focal point of the mirror and the detector was placed at the exit of the mirror to measure the reflected photons from the mirror. Fig. 1(c) shows the simulation results obtained for two scenarios; one assuming Table 1 A comparison of ion beam induced fluorescence counts obtained using different collection systems. Collection system
Fluorescence counts
Ratio
PMT with parabolic mirror PMT
37,979 16,754
2.3
the target holder and sample as non-reflective and the other as reflective. The results are also compared with the existing set-up in which no mirror was present and the detector was placed behind the sample. The maximum and minimum light collection efficiency of parabolic mirror system was projected to be 63% and 24% respectively which is higher than simulated efficiency of the existing set up (17%).
3. Experimental results The existing light collection system was modified by introducing the custom-made parabolic mirror in the set-up. The parabolic mirror was aligned perpendicular to the beam axis and was placed on precision closed loop three axis stage (Mechonics Model MS-030) to move the mirror such that the focal point of the parabolic mirror coincides with the beam focus on the sample. The closed loop stage is controlled by the stage controller: CU30CL. The light-weight closed loop stage was attached to the main stage (open loop three axis stage, PI N-310K059) to which the target holder was mounted. The schematic and the actual set-up is shown in Fig. 2. The closed loop stage has 25 mm translation in all three axis with position accuracy of 50 nm and is chosen as light weight in order to reduce the load on the main stage. The mirror characterization experiments were carried out with a focused 1.6 MeV helium ion beam. The ion beam induced fluorescence was obtained by scanning the focused beam onto the lanthanide doped nano rods (NaYF4: 2% Tm 60% Yb) which were mounted on a 100 nm thick silicon nitride window. These upconversion nanoparticles produce blue and red emission (peaks at 450, 470 and 650 nm) under the 980 nm laser excitation. In this experiment the isotropic emission was collected using the parabolic mirror. The collimated fluorescence light from the parabolic mirror was focused onto a 600 lm diameter fiber using a 2 inch focal length plano-convex lens. The emitted light was guided out of the vacuum chamber through the fiber and was
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076
4
S.K. Vanga et al. / Nuclear Instruments and Methods in Physics Research B xxx (2015) xxx–xxx
Fig. 4. The ion beam induced fluorescence images of lanthanide doped nanorods collected using (a) the custom-made parabolic mirror together with a focusing mirror and PMT and (b) with a PMT placed behind the sample.
connected to the photomultiplier tube (PMT) (Hamamatsu PMT R7401P) for the detection of fluorescence emission. For the characterization of the fabricated custom-made mirror, first the mirror was translated such that the focal point of the parabolic mirror approximately coincides the ion beam focus on the sample. The ion beam was scanned electrostatically in an area of 54 lm by 54 lm and the mirror stage was scanned in Z direction by observing the fluorescence counts from the PMT. A plot of the fluorescence counts as a function of mirror position in Z direction can be seen in Fig. 3(a). The full width at half maximum (FWHM) of the plot gives the depth of the focus (DoF) of mirror which is 80 lm. Once the focal point in Z direction was determined, the stage was moved to the optimal Z position and raster scanned in X and Y directions to find the field of view (FoV) of the mirror. Fig. 3(b) shows the intensity plot of XY scan as a function of fluorescence counts. The FWHM of these plots gives the field of view of the mirror in X and Y directions which is 96 lm and 110 lm respectively. The cell imaging work at our facility requires a scan area of 50 lm by 50 lm which is within the mirror field of view. The simultaneous ion beam induced fluorescence imaging and scanning transmission ion microscopy imaging capability of parabolic mirror was tested by imaging the same lanthanide doped fluorescent nano rods. The STIM detector was translated and positioned behind the mirror hole and ion beam was scanned in an area of 13.2 lm by 13.2 lm. Fig. 3(c) and (d) shows the images of the off-axis STIM and ion beam induced fluorescence obtained simultaneously. To detect sufficient number of fluorescence counts using the system, ion beam charge collection was adjusted so that 165 alpha particles incident on the sample for each pixel in the image. The comparison between the existing collection system and the new parabolic mirror collection system was performed by obtaining the fluorescence images of the same lanthanide doped nanorods. The PMT used in both cases is the same (Hamamatsu PMT R7401P), which has a detection wavelength range is 300– 650 nm. The fluorescence counts was normalized with the STIM counts and the results were tabulated in Table 1. The collected images can be seen in Fig. 4. It is clear that the introduction of parabolic mirror enhances the light collection by a factor of 2.3, though the ray tracing simulation showed a factor of 3.7 improvement. This difference is reasonable since the target holder and the sample is not 100% reflective. Over the time aluminum may oxidize hence reduce the reflectivity so we plan to coat the mirror inner surfaces with a reflective coatings and also a protective coating to avoid oxidation. Future work will be focused on the comparison of ion beam induced fluorescence emission from different fluorescent probes and their radiation stability.
4. Conclusion In conclusion, a custom made parabolic mirror has been designed to improve the collection efficiency of ion beam fluorescence imaging. The custom-made mirror is installed in the current set-up and characterized experimentally to obtain the mirror characteristics such as depth of focus and field of view of the mirror. When compared to the existing collection system, the collection efficiency was improved by a factor of 2.3 by introducing the new parabolic mirror in the system. Adding this component to the set-up not only enhances the fluorescence collection but also allows us to obtain the STIM images simultaneously. Acknowledgement This work is supported by the Ministry of Education - Singapore, Academic Research Fund Tier 2 grant (R-144-000-306-112). The authors would like to acknowledge Xiaogang Liu and Yuhai Zhang from the Department of Chemistry for supplying the lanthanide doped nanorods. References [1] K.G. Malmqvist, M. Elfman, G. Remond, C. Yang, PIXE and ionoluminescence – a synergetic analytical combination, Nucl. Instr. Meth. B 109–110 (1996) 227– 233. [2] J. Zuk, T.J. Ochalski, M. Kulik, J. Lis, A.P. Kobzev, Effect of oxygen implantation on ionoluminescence of porous silicon, J. Lumin. 80 (1999) 187–192. [3] A.A. Bettiol, D.N. Jamieson, S. Prawer, M.G. Allen, Ion beam induced luminescence from a nuclear microbeam from diamond and other crystals, Nucl. Instr. Meth. B 85 (1994) 775–779. [4] J. Pallon, C. Yang, R.J. Utui, M. Elfman, K.G. Malmqvist, P. Kristiansson, K.A. Sjoland, Ionoluminescence technique for nuclear microprobes, Nucl. Instr. Meth. B 130 (1–4) (1997) 199–203. [5] P. Rossi, C.D. Maggio, G.P. Egeni, A. Galligioni, G. Gennaro, L. Giacomelli, A. Lo Giudice, M. Pegoraro, L. Pescarini, V. Rudello, E. Vittone, Cytological and histological structures identification with the technique IBIL in elemental microanalysis, Nucl. Instr. Meth. B 181 (2001) 437–442. [6] F. Watt, A.A. Bettiol, J.A. van Kan, M.D. Ynsa, R. Minqin, R. Rajendran, C. Huifang, S. Fwu-Shen, A.M. Jenner, Imaging of single cells and tissue using MeV ions, Nucl. Instr. Meth. B 267 (12–13) (2009) 2113–2116. [7] R. Norarat, V. Marjomäki, X. Chen, M. Zhaohong, R. Minqin, C.-B. Chen, A.A. Bettiol, H.J. Whitlow, F. Watt, Ion-induced fluorescence imaging of endosomes, Nucl. Instr. Meth. B 306 (2013) 113–116. [8] J.A. van Kan, P. Malar, A.B. de Vera, The second generation Singapore high resolution proton beam writing facility, Rev. Sci. Instrum. 83 (2) (2012) 02B902. [9] F. Watt, X. Chen, C.-B. Chen, C.N.B. Udalagama, J.A. van Kan, A.A. Bettiol, Whole cell structural imaging at 20 nanometre resolutions using MeV ions, Nucl. Instr. Meth. B 306 (2013) 6–11. [10] ZEMAX Optical Design Program: User’s Guide,
Please cite this article in press as: S.K. Vanga et al., Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy, Nucl. Instr. Meth. B (2015), http://dx.doi.org/10.1016/j.nimb.2015.01.076