Dimensional-Grating-Coupled Surface Plasmon ... - OSA Publishing

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Kenji Kintaka1)*, Xiaoqiang Cui2), Keiko Tawa2), and Junji Nishii1,3). 1) Photonics Research Institute, National Institute of Advanced Industrial Science and ...
© 2009 OSA/FiO/LS/AO/AIOM/COSI/LM/SRS 2009 a1738_1.pdf AThA6P.pdf

100-Fold Enhancement of Fluorescence Imaging by TwoDimensional-Grating-Coupled Surface Plasmon Resonance Kenji Kintaka1)*, Xiaoqiang Cui2), Keiko Tawa2), and Junji Nishii1,3) 1) Photonics Research Institute, National Institute of Advanced Industrial Science and technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan 2) Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan 3) Research Institute for Electronic Science, Hokkaido University, Kita 21 Nishi 10, Kita-ku, Sapporo 001-0021, Japan *[email protected]

Abstract: Silver-coated two-dimensional periodic structures were fabricated for high-efficiency excitation of surface plasmon resonance. The fluorescence image of labeled proteins on the periodic structure was 100 times as bright as that on a flat glass plate. ©2009 Optical Society of America OCIS codes: (180.2520) Fluorescence microscopy; (240.6680) Surface plasmons; (050.6624) Subwavelength structures

1. Introduction Surface-plasmon-field-enhanced fluorescence spectroscopy (SPFS) has been investigated as one of promising technologies for detection of week fluorescence from molecules or bio-related materials [1, 2]. As excitation technique of surface plasmon resonance (SPR) for SPFS, a prism coupling method with Kretschmann configuration has been widely used so far due to its simple structure. On the other hand, we have investigated a grating coupling method with surface-relief subwavelength periodic structure for SPR excitation with low incident angle [3-6]. In the previous works, we have demonstrated fluorescence imaging enhanced by SPR with one-dimensional (1-D) periodic structures. In this work, we fabricated two-dimensional (2-D) periodic structures for more sensitive biosensors using higher-efficiency SPR excitation under unpolarized lamp illumination of a conventional fluorescence microscope. We demonstrated fluorescence microscopic imaging of Cy5-labeled proteins on the 2-D periodic structure, which was brighter than that on the 1-D periodic structure, and 100 times brighter than that on a flat glass plate. 2. Fabrication of periodic structures 2-D periodic structures were fabricated on a silica glass substrate. An i-line photoresist (TDMR-AR80, Tokyo Ohka Kogyo Co., Ltd.) was spin-coated and exposed twice to 325-nm-wavelength He-Cd laser light by use of a two-beam interference method. Between the two irradiation steps, the substrate was rotated 90°. As a result, a 2-D periodic holed pattern with 400 nm period was obtained. The 2-D periodic pattern was transferred to the glass substrate with 25 nm depth by a reactive ion etching with mixture gas of C3F8 and Ar. After the residual photoresist was removed, an Ag layer of 200 nm thickness with a Cr adhesion layer was deposited by RF-sputtering on the 2-D periodic structure. Furthermore, a SiO2 layer with 20 nm thickness were deposited on the Ag layer via another Cr adhesion

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Fig. 1. SPM images of the fabricated 2-D periodic structure (a) before and (b) after coating of SiO2 /Cr/Ag/Cr multi-layered film

© 2009 OSA/FiO/LS/AO/AIOM/COSI/LM/SRS 2009 a1738_1.pdf AThA6P.pdf

layer by RF-sputtering in order to suppress the quenching of fluorescence by the Ag layer [2] and to prevent oxidation of the Ag layer. Figure 1 shows the fabricated 2-D periodic structure before and after coating of the SiO2/Cr/Ag/Cr multi-layer film observed by scanning probe microscope (SPM). The sidewall of each hole was slanted slightly and surface roughness was increased by the film coating. 3. Measurement of SPR and SPFS characteristics Cy5-labeled proteins were stabilized on the surface of the fabricated 2-D periodic structure in a phosphate buffer saline solution for fluorescence measurement. Figure 2 illustrates a schematic view of an experimental setup for SPR and SPFS characteristics. A He-Ne laser light with 633 nm wavelength passing through a depolarizer was used as a light source. The reflection light was measured by a photodiode, and the fluorescence light was detected by a photomultiplier through a band-pass filter of 670±5 nm wavelength at a angle of 55° with the incident beam. Figure 3(a) shows the relationship between the incident angle θ and the reflectivity with substrate rotation angle φ of 0, 30, 60, and 90 degree. The substrate rotation angle φ of 0 degree means that the incident plane is parallel to one of the grating vectors of the 2-D periodic structure. The reflectivity dips caused by SPR excitation were clearly observed at the incident angle θ of 6.5°, 7.7° and 15°, 7.3° and 18.5°, and 6.7° for the rotation angle φ of 0, 30, 60, and 90 degree, respectively. These results agree well with the theoretical prediction of the incident-angle dependence calculated by a commercially available software (GSOLVER 4.2, Grating Solver Development Co.) based on rigorous coupled wave analysis (RCWA). The slight difference of the SPR angles between φ=0° and φ=90° or between φ=30° and φ=60° was probably caused by measurement accuracy in the rotation angle and asymmetry in the fabricated structure. Figure 3(b) shows the incident angle dependence of the fluorescence intensity with

Fig. 2. Experimental setup for SPR and SPFS measurements

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Fig. 3. Incident angle dependence of (a) reflectivity and (b) fluorescence intensity with substrate rotation angle φ of 0, 30, 60, and 90 degree

© 2009 OSA/FiO/LS/AO/AIOM/COSI/LM/SRS 2009 a1738_1.pdf AThA6P.pdf

substrate rotation angle φ of 0, 30, 60, and 90 degree. The intensity peaks were observed at the same incident angles as those of SPR dips in any rotation angles φ. It is evident that Cy5 was efficiently excited by SPR field coupled with the incident He-Ne laser light by the 2-D periodic structure. 4. Fluorescence imaging under microscope Fluorescence microscopic imaging was carried out by using a commercially available optical microscope (BX51WI, Olympus Co.) with a halogen lamp and a 10X objective (NA=0.30). Figure 4(a)-(d) show fluorescence images of Cy5-labeled proteins on the fabricated 2-D periodic structure, a 1-D periodic structure with the same period and grating depth as the 2-D structure, a SiO2/Cr/Ag/Cr-multilayer-film-coated flat glass plate, and an uncoated flat glass plate, respectively. The illumination condition was kept constant for all the imaging. The fluorescence intensity after background subtraction on the 2-D periodic structure was 3 times, 25 times, and 100 times as high as those on the 1-D periodic structure, the coated flat glass plate, and the uncoated flat glass plate, respectively. It is evident that the 2-D periodic structure can efficiently couple between SPR and the unpolarized incident light, while the 1-D periodic structure can only couple between SPR and the p-polarized light parallel to the grating vector in the incident light.

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Fig. 4. Fluorescence microscope images of Cy5-labeled proteins on (a) 2-D periodic structure, (b) 1-D periodic structure, (c) SiO 2/Cr/Ag/Crmultilayer-film-coated glass plate, and (d) uncoated glass plate

5. Conclusions We have designed and fabricated an Ag-coated 2-D periodic structure for SPR excitation with an unpolarized lamp of fluorescence microscope. The incident angles for SPR excitation have agreed well with the theoretically predicted values. The enhanced fluorescence microscopic imaging of Cy5-labeled proteins has been demonstrated by using a conventional optical microscope. The fluorescence image on the 2-D periodic structure was 100 times brighter than that on the uncoated flat glass plate. Further enhancement of the fluorescence would be realized by optimization of the periodic structures. Acknowledgements This work was supported by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas “Strong PhotonMolecule Coupling Fields (No. 470),” No. 19049016, from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 6. References [1] T. Liebermann, and W. Knoll, “Surface-plasmon field-enhanced fluorescence spectroscopy,” Colloids and Surfaces 171, 115-130 (2000). [2] K. Tawa, and K. Morigaki, “Substrate supported phospholipids membranes studied by SPR and surface plasmon fluorescence spectroscopy (SPFS),” Biophys. J. 89, 2750-2758 (2005). [3] K. Tawa, H. Hori, K. Kintaka, K. Kiyosue, Y. Tarsu, and J. Nishii, “Optical microscopic observation of fluorescence enhanced by gratingcoupled surface plasmon resonance,” Opt. Express 16, 9781-9790 (2008). [4] H. Hori, K. Tawa, K. Kintaka, J. Nishii, and Y. Tatsu, “Influence of groove depth and surface profile on fluorescence enhancement by grating-coupled surface plasmon resonance,” Opt. Rev. 16, 216-221 (2009). [5] N Akashi, K. Tawa, Y. Tatsu, K. Kintaka, and J. Nishii, “Application of grating substrate fabricated by nanoimprint lithography to surface plasmon field-enhanced fluorescence microscopy and study of its optimum structure,” Jpn. J. Appl. Phys. 48, 062002 (2009). [6] N Akashi, K. Tawa, Y. Tatsu, K. Kintaka, and J. Nishii, “Grating substrate fabricated by nanoimprint lithography for fluorescence microscopy,” Jpn. J. Appl. Phys. 48, 06FH17 (2009).