Parallel scan spectral surface plasmon resonance imaging
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1 Parallel scan spectral surface plasmon resonance imaging Le Liu,* Yonghong He, Ying Zhang, Suihua Ma, Hui Ma, and Jihua Guo Laboratory of Optical Imaging and Sensing, Graduate School at Shenzhen, Tsinghua University, Shenzhen, , China *Corresponding author: Received 28 May 2008; revised 17 September 2008; accepted 17 September 2008; posted 18 September 2008 (Doc. ID 96605); published 15 October 2008 We describe a parallel scan spectral surface plasmon resonance (SPR) imaging technique. We demonstrate experimentally, with a line-shaped light illumination, that an image acquired with an area CCD detector provides both SPR wavelength information and one-dimensional spatial distribution. Thus two-dimensional distribution of the refractive index of the entire sensing plane can be obtained with a one-dimensional optical line parallel scan. The technique offers advantages of both high sensitivity and high throughput, and could have potential applications in biochips analysis Optical Society of America OCIS codes: , , Introduction Surface plasmon resonance (SPR) sensing has been widely used in biology and chemistry research [1]. This label-free sensing approach offers an advantage of extremely high sensitivity [2]. The use of SPR point sensing is rapidly gaining acceptance in diverse fields, such as gene detection, protein interactions investigation, and chemical reactions monitoring [3]. O Brien et al. [4] reported one-dimensional SPR imaging using the angle interrogation, where a camera was used to record both one-dimensional spatial position information and SPR angle profiles. However, speckle effect caused by the monochromatic light source, which reduces the imaging quality, is difficult to cancel even with a rotating diffuser. Fu et al. [5] reported one-dimensional SPR imaging using the wavelength interrogation, where a linear variable filter was used to generate light with wavelength distributions in one spatial dimension. The wavelength resolution is limited by the bandpass function of the linear filter, resulting in a limitation of refractive index resolution (7: refractive index unit, RIU) /08/ $15.00/ Optical Society of America Recently, with the appearance of biochips, such as protein arrays and gene arrays, it is essential to develop an appropriate two-dimensional SPR technique with high throughput to make gene or protein screening. Rothenhäusler et al. reported a surface plasmon microscopy (SPM) technique with high sensitivity and resolution [6]. However, the technique offers a narrow dynamic range and qualitative sensing, rather than quantitative sensing, as the measured reflection intensity does not linearly correspond to the refractive index, and two different refractive indices may correspond to the same reflection intensity. Yuk et al. reported two-dimensional point scan SPR imaging, where the intensity of each pixel linearly corresponds to the refractive index [7 9]. The point-by-point scanning method is too timeconsuming to be suited for high-throughput analysis of biochips. Otsuki et al. reported wavelength scan spectral SPR imaging, where multiple pictures were used to calculate the refractive index of each sensing point [10]. The variation of CCD imaging, e.g., noise, could reduce sensitivity. Moreover, acquired images could have distortions due to a different depth of the focus from the prism. In this paper, we present a parallel scan SPR imaging method, for the first time as far as we know. The 5616 APPLIED OPTICS / Vol. 47, No. 30 / 20 October 2008
2 Fig. 1. Schematic of a parallel scan spectral SPR imaging device: A, white light point source; B, achromatic convex lens; C, polarizer; D, cylindrical convex lens; E, Kretschmann type SPR module (E1, prism; E2, gold film, where a refractive index distribution model of different concentrations of glucose solution is arranged on the gold film; E3, one-dimensional translation stage); F, G, H, I, spectrometers; J, CCD camera. novelty of the method is that we combine wavelength interrogation SPR with parallel one-dimensional scanning to quickly sense a two-dimensional area without speckle effect. We demonstrate experimentally, with a line-shaped light illumination, that an image acquired with area CCD detector provides both SPR wavelength information and a onedimensional spatial distribution. Thus a twodimensional distribution of the refractive index of the entire sensing plane can be obtained with a one-dimensional optical line parallel scan. 2. Experimental Setup Figure 1 shows the schematic of the experimental system used in this study. A halogen lamp with a power of 100 W is condensed into a white light point source. The light is collimated with an achromatic convex lens (f ¼ 50 mm) and then polarized with a linear glass polarizer. After passing through a cylindrical convex lens (f ¼ 40 mm), the light is focused into a narrow line shape to irradiate the SPR module. The SPR module is configured in a Kretschmann manner with an attenuated total reflection method: a right angle prism (SF6 glass, RI ¼ 1:78) is coated with a 38 nm gold film on the hypotenuse plane to produce SPR phenomena. The prism is fixed on a one-dimensional translation stage that allows the sensing layer to move in the plane of the sample orthogonal to the incident line-shaped beam. Thus we can scan the sensing area with the line-shaped beam by moving the one-dimensional translation stage. The incident line-shaped beam is focused onto the gold film with a central incident angle of approximately 51:7. The reflected beam goes into the entrance slit of a commercial spectrometer with a 300 line=mm grating and a 20 μm slit (SpectralPro 150, Acton research). The 20 μm entrance slit, which is placed 10 cm away from the line-shaped beam, permits only the light whose incident angle is in a small range (51:70:005 ) to enter the spectrometer. Thus we have an approximately fixed incident angle. The light leaving the spectrometer is collected with a CCD camera (Q-imaging RETIGA EXi 1394) placed on the focal plane. A personal computer controls the CCD to acquire digitized images with a dynamic range of 12 bits and a resolution of pixels. We adjust the polarizer in the input light path (C in Fig. 1) and capture two images when the incident light is p-polarized and s-polarized, respectively. Figure 2(a) shows one row of each image; the darker curve A is from the p-polarized image, the lighter one B is from the s-polarized image. In order to compensate a spectral response of components such as a light source or a CCD, we divide each row of the p- polarized image by the corresponding row in the s- polarized image to produce a new image. A typical row of the image is shown in Fig. 2(c) (lighter curve). Because the diffraction efficiency of the grating is strongly dependent on the polarization, we examine Fig. 2. (a) Darker curve A is a plotted row from the p-polarized image; the lighter curve B is the corresponding row from the s-polarized image. (b) The measured ratio of the grating efficiency for s polarization and for p polarization. (c) the lighter curve C is A divided by B in (a); the darker curve D is C divided by the curve in (b) and is thus the SPR spectrum (jr p ðλþj 2 =jr s ðλþj 2 ). 20 October 2008 / Vol. 47, No. 30 / APPLIED OPTICS 5617
3 Fig. 3. (a) Refractive index distribution model. (b) A typical image captured by CCD after a compensating spectral response of the system. Each row represents the SPR spectrum jrp ðλþj2 =jrs ðλþj2 of one point in the sensing plane. The darker bands represent the glucose solution droplets. The white bands represent the air region. (c) The intensity of three typical rows from each darker band in (b). the spectral response of the system for the p and s polarizations using a bare gold film thick enough not to cause extinction due to SPR. The ratio of grating efficiency for s and p polarization is plotted in Fig. 2(b). Then we divide the lighter curve C in Fig. 2(c) by the experimental curve in Fig. 2(b) to obtain the darker curve D in Fig. 2(c) to compensate for the diffraction efficiency of the grating. The intensity of the final curve is an SPR spectrum (jrp ðλþj2 = jrs ðλþj2, where Rp and Rs are the reflectance of the ppolarized light and s-polarized light, respectively) corresponding to one point in the illuminated line of a sensing plane. With the wavelength given by the minimum intensity, which is called SPR wavelength, we could work out the refractive index of one sensing point from one row according to Fresnel s equations [11] and integration over a 0:01 range of angle. Therefore we could obtain the refractive indices of one line area of the sensing plane from one image. Moving the translation stage to scan the sensing plane, a series of p-polarized images are acquired with the CCD camera. We use only one spolarized image for spectral response compensating calculation instead of all s-polarized images to simplify the sensing progress. As each calculated image contains the refractive index information of one line, we could work out a quantificational refractive index distribution of a whole sensing plane from the 200 images. With this parallel scan SPR imaging setup, we can sense an area of 8 mm 8 mm in 60 s, including the data processing time of 30 s. When we adjust the system with a bare gold film, i.e., with homogeneous medium (water or air) to be tested, we tune optical components to obtain the minimal standard deviation of refractive index of the whole sensing plane to ensure that the incident line-shaped beam is straight and that it is orthogonal to the incident plane. 3. Results and Discussion A glucose solution was used in refractive index sensing experiments. The relationship between 5618 APPLIED OPTICS / Vol. 47, No. 30 / 20 October 2008 refractive index n and concentration C is n ¼ 1:325 þ 1: C, where C is concentration in grams per liter [12]. To test the imaging capability of the method, we developed a sensing model (shown in Fig. 3(a)) by manually dropping nine droplets (3 3 array) of glucose solution with different concentrations onto the gold film. The diameter of each glucose droplet was approximately 2:0 mm, and the distances Fig. 4. (a) Image of refractive index distribution of the sensing model, with nine glucose solution droplets of three different concentrations: 0 g=l, 2 g=l, 4 g=l, where corresponding refractive indices are , , The distribution is the following: first row, 0 g=l, 2 g=l, 4 g=l; second row, 2 g=l, 4 g=l, 2 g=l; third row, 4 g=l, 2 g=l, 0 g=l. (b) The plotted dashed row in (a), which is across the glucose solution droplets. The RI of the background (air RIU) is out of the sensing range ( in this experiment), so the intensity of this area is set to minimum.
4 Fig. 5. (a) Sensing image of a gold film with air medium; the standard deviation of SPR wavelength is 0:38 nm. Experimental conditions are the following: BK7 glass prism, 2 nm Cr film, 40 nm gold film, sensing target is air. (b) Plotted row A of (a). (c) Plotted column B of (a). between them varied from 0:2 mm to 0:9 mm. The concentrations of 0 g=l, 2 g=l, and 4 g=l corresponded to the refractive indices of these droplets of , , and , respectively. Letting the illuminated line area cross first column of the 3 3 array, we captured a typical sensing image with the CCD camera. Figure 3(b) shows the image after being compensated with the system spectral response curve for different wavelengths. With each row representing the spectrum of one certain point in the line area, the entire picture of Fig. 3(b) is the spectrum of the illuminated line area. The three darker bands are the spectrum of the area of glucose solution droplets. The intensity of a typical row from each band is shown in Fig. 3(c). We make each five rows averaged and 10th order polynomial curve fitting to reduce noise. The SPR wavelengths of the three droplets are found to be 793:5 nm, 794:9 nm, and 796:3 nm. We can see from Fig. 3(c) that the SPR resonance wavelengths of the three rows have a difference of 1:4 nm, which correspond to a refractive index unit (RIU) difference of three droplets. Scanning the sensing model with the translation stage for 200 steps with an interval of 40 μm, a array of SPR wavelength is obtained. From the array, we finally obtained the quantificational refractive indices distribution map of the sensing model as shown in Fig. 4(a). A typical row across the droplets is plotted in Fig. 4(b). The refractive index of the background (air RIU) is out of the sensing range ( in this experiment), so the intensity of this area is set to minimum. In Fig. 4(a), the shape of nine droplets is clearly observed, and the intensity of each pixel linearly represents the refractive index of the corresponding point in the sensing plane. It is also shown that the sensing plane is out of focus to some extent. This could be improved by decreasing the size of the point light source or by selecting a cylindrical lens with optimal focal length. To get the spatial stability of the SPR wavelength of our system, a gold film with an air medium was measured as shown in Fig. 5(a). The SPR wavelength of a typical row A and column B were plotted in Fig. 5(b) and Fig. 5(c), respectively. The standard deviation of SPR wavelength is calculated to be 0:38 nm. Figure 6 shows the SPR wavelength variation with time within 2 hours; the standard deviation is measured to be 0:086 nm. To evaluate sensing resolution of our system, we use the formula reported in Ref. [13]: δn ¼ δλ S n ; where δλ is the resolution in SPR wavelength, and S n is the sensitivity of the SPR sensor. In our system, S n ¼ 1:4 nm=0:0003 RIU ¼ 4667 nm=riu and δλ ¼ 0:38 nm. So with this method, resolution of our system is calculated to be 8: RIU, which is better than 7: RIU in Fu et al. s report [5], comparable with 7: RIU in Yuk et al. s report [7], and sensitive enough to analyze protein interactions on protein arrays [7]. Fig. 6. Variation of SPR wavelength of a fixed point in a sensing plane within 2 hours; the standard deviation of SPR wavelength is 0:086 nm. Experimental conditions are the following: BK7 glass prism, 2 nm Cr film, 40 nm gold film, sensing target is air. 20 October 2008 / Vol. 47, No. 30 / APPLIED OPTICS 5619
5 Fig. 7. (a) 2-D SPR wavelength map of a gold film scratched by a needle tip. Experimental conditions are the following: BK7 glass prism, 2 nm Cr film, 40 nm gold film, sensing target is air. (b) The enlarged marked area in (a). (c) Partially plotted column A of (b). (d) Partially plotted row B of (b). (e) A microscopic picture of the marked square area in (b). To test the spatial resolution of the system, a needle tip is used to scratch the sensing plane to get an ultrathin line on the gold film. Figure 7(a) shows a whole image of the scratched gold film obtained with our system. The marked area in Fig. 7(a) is enlarged as shown in Fig. 7(b). Column A and row B are partially plotted in Fig. 7(c) and 7(d), respectively. It is shown from Fig. 7(c) and 7(d) that the full widths at half-maximum (FWHM) of the scratched line in both transverse and lengthways orientations are within one pixel, which corresponds to a spatial distance of 40 μm. Figure 7(e) is the microscopic picture (objective 40, XPL-1530, Yuexian Company, China) of the marked square area in Fig. 7(b), we can see that the widths of the scratched line in transverse and in lengthways orientations are 33 μmand18 μm, respectively. So the spatial resolution of the system is smaller than the spatial distance of one pixel, i.e., better than 40 μm. The resolution is comparable with results from other studies, e.g., 40 μm in O Brien et al. s report [4], and enough for biochip analysis (typical point size 100 μm or larger). 4. Conclusion In conclusion, we have demonstrated that the parallel scan spectral SPR imaging technique could successfully analyze the distribution of refractive indices of the sensing plane. This technique has a refractive index resolution of 8: RIU and a spatial resolution of better than 40 μm in both transverse and lengthways orientations. As a kind of wavelength interrogation based SPR method, it has the advantage of being label-free and having as high a sensitivity as the other SPR technique. Besides, because of using a line-shaped beam to sense a line in one time, parallel scanning to sense the whole plane, this new parallel scan SPR imaging technique has a high throughput. The distribution of refractive indices of the sensing plane can be obtained verifiably and quantitatively. This method could have potential applications in biochips analysis. This research was made possible with the financial support from the 863 project, China (2006AA06Z402) and NSFC, China, for the projects and References 1. J. Homola, S. S. Yee, and G. Gauglitz, Surface plasmon resonance sensors: review, Sens. Actuators B 54, 3 15 (1999). 2. Z. L. Sun, Y. H. He, and J. H. Guo, Surface plasmon resonance sensor based on polarization interferometry and angle modulation, Appl. Opt. 45, (2006). 3. J. Homola, Present and future of surface plasmon resonance biosensors, Anal. Bioanal. Chem. 377, (2003). 4. M. J. O Brien II, V. H. Perez-Luna, S. R. J. Brueck, and G. P. Lopez, A surface plasmon resonance array biosensor based on spectroscopic imaging, Biosen. Bioelectron. 16, (2001). 5. E. Fu, S. Ramsey, R. Thariani, and P. Yager, One-dimensional surface plasmon resonance imaging system using wavelength interrogation, Rev. Sci. Instrum. 77, (2006). 6. B. Rothenhäusler and W. Knoll, Surface-plasmon microscopy, Nature 332, (1988). 7. J. S. Yuk, S. J. Yi, H. G. Lee, H. J. Lee, Y. M. Kim, and K. S. Ha, Characterization of surface plasmon resonance wavelength by changes of protein concentration on protein chips, Sens. Actuators B 94, (2003). 8. J. S. Yuk, S. H. Jung, J. W. Jung, D. G. Hong, J. A. Han, Y. M. Kim, and K. S. Ha, Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor, Proteomics 4, (2004). 9. J. W. Jung, S. H. Jung, H. S. Kim, J. S. Yuk, J. B. Park, Y. M. Kim, J. A. Han, P. H. Kim, and K. S. Ha, Highthroughput analysis of GST-fusion protein expression and activity-dependent protein interactions on GST-fusion protein 5620 APPLIED OPTICS / Vol. 47, No. 30 / 20 October 2008
6 arrays with a spectral surface plasmon resonance biosensor, Proteomics 6, (2006). 10. S. Otsuki, K. Tamada, and S. Wakida, Wavelength-scanning surface plasmon resonance imaging, Appl. Opt. 44, (2005). 11. H. Raether, Surface Plasmons on Smooth and Rough Surfaces and on Gratings (Springer, 1988). 12. J. S. Maier, S. A. Walker, S. Fantini, M. A. Fanceschini, and E. Gratton, Possible correlation between blood-glucose concentration and the reduced scattering coefficient of tissues in the near-infrared, Opt. Lett. 19, (1994). 13. J. Homola, On the sensitivity of surface plasmon resonance sensors with spectral interrogation, Sens. Actuators B 41, (1997). 20 October 2008 / Vol. 47, No. 30 / APPLIED OPTICS 5621
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