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Abstract
We present for the first time a surface plasmon-enhanced dark-field microsphere-assisted microscopy in imaging both low-contrast dielectric objects and metallic ones. We demonstrate, using an Al patch array as the substrate, the resolution and contrast in imaging low-contrast dielectric objects are improved compared to that of the metal plate substrate and a glass slide in dark-field microscopy (DFM). 365-nm-diameter hexagonally arranged SiO nanodots assembled on the three substrates can be resolved, with the contrast varied from 0.23 to 0.96, and the 300-nm-diameter hexagonally close-packed polystyrene nanoparticles can only be discerned on the Al patch array substrate. The resolution can be further improved by using the dark-field microsphere-assisted microscopy, and an Al nanodot array with a nanodot diameter of ∼65 nm and a center-to-center spacing of 125 nm can be just resolved, which cannot be distinguished in a conventional DFM. The focusing effect of the microsphere, as well as the excitation of the surface plasmons, provides evanescent illumination with enhanced local electric field (E-field) on an object. The enhanced local E-field acts as a near-field excitation source to enhance the scattering of the object, resulting in the improvement of imaging resolution.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
The development of optical microscopy has greatly accelerated the pace of our exploration to the micro-world. However, the lateral resolution of a traditional optical microscope is limited to λ/2 (where λ is the wavelength of illumination) due to the loss of evanescent waves that carry the high-spatial frequency information of an object exists in the near-field [1]. Various techniques have been developed to collect the evanescent waves, including scanning near-field optical microscopy [2,3], superlens [4,5], hyperlens [6], microsphere-assisted microscopy [7–15], structured illumination microscopy [16], plasmonic structured illumination microscopy [17,18], localized plasmonic structured illumination microscopy [19,20], etc. Compared with other super-resolution techniques, the microsphere-assisted microscopy has the advantages of label-free, direct observation, white light illumination and so on.
In microsphere-assisted microscopy, researchers usually conduct experiments under bright-field illumination (BFI). However, it is still difficult to observe label-free, low-contrast specimens using a microsphere-assisted bright-field microscope. Recently, Zhou et al. found that the imaging contrast and uniformity were improved when applying dark-field illumination (DFI) rather than BFI in microsphere-assisted microscopy [21]. Perrin et al. demonstrated that transparent samples, having detail sizes of a few hundred nanometers, could be observed by using the microsphere-assisted dark-field technique in transmission mode [22]. The advantage of DFI is that only the light scattered or diffracted by samples enters the objective lens, which can effectively reduce the influence of background signals and improve the signal-to-noise ratio. However, the imaging resolution is still diffraction limited. Meanwhile, studies have demonstrated that the excitation of plasmons in metallodielectric nanostructures improves the spatial resolution and the contrast in microsphere-assisted microscopy. Brettin et al. demonstrated that by using Au nanoplasmonic arrays as the substrate, the resolution in imaging fluorescent objects could reach ∼λ/7. The observed super-resolution was based on the emission of the objects coupled to the localized surface plasmon resonances (LSPRs) in the Au nanodisc arrays [23]. We experimentally demonstrated that the surface plasmon polaritons (SPPs) excited on the surface of the plasmonic objects or plasmonic substrates could improve the imaging resolution in microsphere-assisted bright-field microscopy [24,25]. Pei et al. used a metal/dielectric multilayer coated grating as the substrate, polystyrene (PS) nanoparticles with a diameter of 200 nm and a center-to-center spacing of 300 nm could be resolved by barium titanate glass microsphere-assisted microscopy. The above proposed plasmonic substrates enhance the resolution in microsphere-assisted microscopy under BFI, but the signal-to-noise is low in a bright-field optical microscope.
To overcome the challenges of limited resolution under DFI and low signal-to-noise ratio by using plasmonic substrate under BFI for imaging low-contrast dielectric objects in microsphere-assisted microscopy, in this study, we combine the two techniques effectively, and present surface plasmon-enhanced dark-field microsphere-assisted microscopy for the first time. The imaging resolution and contrast are improved while the low-contrast dielectric objects are assembled on the Al patch array (sub1) instead of on a 50-nm-thick Al film (sub2) or a glass slide (sub3) in dark-field microscopy (DFM). The excitation of SPPs and LSPRs on sub1 provides plasmonic near-field illumination which increases the scattered signal of the sample. Besides, DFI enhances the contrast, thus improving the imaging contrast and resolution. The resolution can be further improved by using the dark-field microsphere-assisted microscopy. A hexagonally close-packed 300-nm-diameter PS nanoparticle array on sub2 can be resolved clearly, and an Al metallic nanodot one with a diameter of ∼65 nm and a center-to-center spacing of 125 nm on sub1 can be just resolved, while they cannot be discerned in an ordinary DFM. We propose that the enhanced evanescent field illumination is generated due to the focusing effect of the microsphere lens, and the excitations of surface plasmons. The enhanced E-field increases the scattering signal of the object, and more scattering signals of the object can be received by the microsphere, which improves the resolution and contrast of dark-field microsphere-assisted microscopy.
2. Experimental
Figure 1(a) illustrates the schematic of the microsphere-assisted microscopy setup. Optical images observed through the SiO2 microsphere were obtained by a Zeiss reflective microscope equipped with a 100× (NA = 0.9) microscope objective, in which the illumination modes can be easily switched between bright-field and dark-field. The light source is a white light halogen lamp with a central wavelength of 540 nm. Under DFI, the incident light with an oblique angle of 63° was focused on the samples. A 6-µm-diameter SiO2 microsphere fully immersed in a polydimethylsiloxane (PDMS) film was in contact with an object to obtain a magnified virtual image of the object.
Fig. 1. (a) Schematic of the experimental setup. (b-1) Optical image of a square Al patch array substrate, the patch diameter is 6 µm, and the center-to-center spacing is 9 µm. (b-2)–(b-4) SEM images of a hexagonally close-packed 300-nm-diameter PS nanoparticle array (sample A); a hexagonal SiO nanodot array with a nanodot diameter of ∼365 nm and a center-to-center spacing of 450 nm (sample B); an Al nanodot array with a nanodot diameter of ∼65 nm and a center-to-center spacing of 125 nm (sample C).
The stencil mask method was used to fabricate a square Al patch array on a glass slide [26]. First, a free-standing SU-8 stencil mask with a square lattice of circular holes was closely attached to a glass slide. Then, a 50-nm-thick Al was deposited through the stencil mask by thermal evaporation, forming a square Al patch array on the glass slide. The Al patch array has a patch diameter of 6 µm and a center-to-center spacing of 9 µm, as shown in Fig. 1(b-1). The Al patch array was used as a substrate (sub1) to place samples. To investigate the effect of the substrate on imaging, we also prepared a glass slide coated with a 50-nm-thick Al film (sub2) and a glass slide (sub3) for comparison.
In the experiment, we prepared three types of samples. Sample A is a hexagonally close-packed 300-nm-diameter PS nanoparticle array, as shown in Fig. 1(b-2). The PS nanoparticle array was assembled on three kinds of substrates by a self-assembly method. Sample B is a hexagonal SiO nanodot array with a nanodot diameter of ∼365 nm and a center-to-center spacing of 450 nm. Sample C is a hexagonal Al nanodot array with a center-to-center spacing of 125 nm and nanodot diameter of ∼65 nm. Samples B and C were prepared on the three substrates by the anodic aluminum oxide (AAO) template method, as shown in Fig. 2. Four steps were involved in the fabrication procedure of the two samples. Firstly, prepare the substrate. Secondly, attach the AAO film closely to the substrate. Then, deposit a 50-nm-thick SiO film or a 50-nm-thick Al film into the AAO template by thermal evaporation. At last, remove the AAO film with a tape, leaving an array of SiO or Al nanodots on the substrate. Figures 1(b-3) and 1(b-4) are SEM images of samples B and C, respectively.
3. Results and discussion
We first compare the images of a monolayer 300-nm-diameter PS nanoparticle array on three types of substrates under BFI and DFI. Figures 3(a), 3(b) and 3(c) show the images of the nanoparticle arrays on sub1, sub2 and sub3 under DFI, respectively. As shown in the figures, the light intensity at the edge of the Al patch is stronger, and the nanoparticle array on sub1 can be resolved (Fig. 3(a)), while the arrays on the other two substrates cannot be resolved at all (Figs. 3(b) and 3(c)). The upper right inserts show the images of the same nanoparticle arrays under BFI. None of the nanoparticle arrays on the three substrates can be discerned by a conventional optical microscope under BFI.
Fig. 3. Images of sample A on sub1 (a), sub2 (b) and sub3 (c) under DFI. The upper right insets show the images of sample A under BFI. The scale bar is 2 µm.
Figures 4(a) and 4(b) present optical images of sample B under the BFI (a) and the DFI (b) by a conventional optical microscope. The top row, Figs. 4(a-1)–4(a-3), illustrates the images of a SiO nanodot array assembled on sub1, sub2 and sub3 obtained under BFI, respectively. Figures 4(b-1)–4(b-3) (the middle row) illustrate the images of a SiO nanodot array on sub1, sub2 and sub3 under DFI, respectively, and the SiO nanodot arrays on the three substrates can be resolved. Directly observed, the images obtained under DFI are significantly clearer than those obtained under BFI. The details of the SiO arrays are better presented under DFI. In addition, we also observed a sample C (Al nanodots) with a diameter of ∼65 nm and a center-to-center spacing of 125 nm, as shown in Fig. 4(c), where the Al nanodots on the three substrates cannot be resolved due to the small size of the sample. Figures 4(d) and 4(e) plot the normalized light intensity profiles of two adjacent nanodots along the dotted lines in Figs. 4(a-1)–4(a-3) and Figs. 4(b-1)–4(b-3). The image contrast is defined as C = (Imax-Imin)/(Imax + Imin), where Imax and Imin are the maximum and minimum intensities, respectively. The calculated contrasts of the images are 0.63, 0.41, 0.20 in Figs. 4(a-1)–4(a-3) and 0.96, 0.66, 0.23 in Figs. 4(b-1)–4(b-3), respectively. It demonstrates that the imaging contrasts are enhanced under DFI, with the contrast is the highest when the sample is placed on sub1.
Fig. 4. (a-1)-(a-3) Optical images of sample B on sub1, sub2 and sub3, respectively, under BFI. (b-1)-(b-3) Optical images of sample B on sub1, sub2 and sub3, respectively, under DFI. (c-1) -(c-3) Optical images of sample C on the three types of substrates under DFI. (d) and (e) Normalized intensity profiles of two adjacent nanodots along the dotted lines in (a-1)-(a-3) and (b-1)-(b-3), respectively. The scale bar is 2 µm.
From the above experiments, it can be concluded that it is more effective to observe the nanostructures in the DFI mode. In our experiments, we also find that there is a difference in the resolution and the contrast when imaging samples placed on different substrates, with the highest contrast and the ability to resolve transparent PS nanoparticle arrays of 300 nm in diameter when the sample is placed on the metallic patch substrate (sub1). We propose that the difference in the resolution and the contrast under DFI is due to the excitation of SPPs.
To investigate the mechanism of resolution and contrast improvement in surface plasmon-enhanced DFM, we use 2D-FDTD to calculate the E-field distributions of a PS nanoparticle array and a SiO nanodot one placed on the three types of substrates. As shown in Figs. 5(c)–5(f), the x-axis direction is the direction where the object is located, and the direction perpendicular to the object plane is the y-axis direction. The Bloch boundary condition is used for the x-direction, and the perfectly matched layer boundary is used for the y-axis direction. Two p-polarized plane waves incident from the top of the sample with an oblique angle of 63°. The mesh size is 2 nm. The refractive index of PS and SiO is 1.6 and 1.55, respectively. The diameter of PS nanoparticles is 300 nm, and that of SiO nanodots is 365 nm, with a center-to-center spacing of 450 nm. The dielectric constant of Al is taken from Palik’s data. The purple line in Figs. 5(c) and 5(e) represents sub1, and the left and the right sides of sub1 are sub3. The orange line in Figs. 5(d) and 5(f) represents sub2. We first calculate the E-field intensity of sample A and sample B on sub1(red line), sub2(green line), and sub3(blue line) at the positions indicated by triangles in Figs. 5(c)–5(f) versus the incident wavelength, as shown in Figs. 5(a) and 5(b). The E-field intensity on sub1 is much stronger than that on sub2 throughout the visible range, and the E-field intensity on sub3 is the weakest. Then, the E-field distributions of sample A and sample B on sub1/sub3 and sub2 at the peak of their E2 maximum are plotted in Figs. 5(c), 5(d) and Figs. 5(e), 5(f), respectively. The right inserts are the zoom-in images of the regions defined by the white-dashed rectangles. As shown in Figs. 5(c) and 5(e), the E-field intensity is weak when the samples are assembled on sub3. However, by placing the dielectric samples on a metallic substrate, whether it is sub1 (Al patch array) or sub2 (Al film), the E-field is locally enhanced between the dielectric sample and the metal substrate, as well as at the gap of the dielectric sample. When sub1 is used, the local E-field is further enhanced. For comparison, 3D-FDTD is used to simulate the surface E-field distributions of a 6 µm*6 µm Al patch array. The wavelength of the incident light is λ=540 nm and the oblique angle of incident light is set to 63°, as shown in Fig. 5(g). The E-field intensity at the edge of the patch array is stronger than that on the patch surface.
Fig. 5. (a), (b) E-field intensity of sample A (a) and sample B (b) on sub1(red line), sub2(green line), and sub3(blue line) at the positions indicated by triangles in (c)-(f) versus the incident wavelength. (c), (d) E-field distributions of sample A on sub1/sub3 (c) and sub2 (d) at the peak of their E2 maximum. (e), (f) E-field distributions of sample B sub1/sub3 (e) and sub2 (f) at the peak of their E2 maximum. The insets are zoom-in images of the regions defined by the white-dashed rectangles. (g) E-field distributions of a 6 µm*6 µm Al patch substrate.
When a light wave incident at the metal/dielectric interface, SPPs can be excited due to the coupling of incident light and collective oscillations of free electrons, accompanied by a strong near-field enhancement. The samples used in the experiments are hexagonally arranged, periodic arrays, both for the PS nanoparticle array and the SiO/ Al nanodot one.
For a hexagonal array, SPPs are excited when their momentum ${k_{spp}}$ matches the momentum of the incident light and the periodic structure, that is:
where ${k_{spp}}$ is the plasmon wave vector, ${k_0}$ is the incident light wave vector, D is the periodicity, n is the refractive index of the dielectric, $\theta $ is the incident angle, and p, q are integers (typically refer to the primary SPP mode, p = 1; q = 0) [27,28]. When the incident light is obliquely incident, SPPs are excited at the interface between the dielectric sample and the metal, exhibiting a strong E-field enhancement at the interface of the metal and the gap of the dielectric sample, especially when using sub1 as substrate. Figure 5(g) reveals that the E-field intensity at the edge of the patch is stronger than that on the patch surface, which is also in good agreement with the experimentally observed brighter edge of the patch. Therefore, we propose that the main reason for a higher resolution and contrast when imaging samples on sub1 is the excitation of SPPs and LSPRs. When polarized light hits the plasmonic patch array, it creates evanescent field which illuminates objects in the near-field, and the details of the objects can be better presented [29]. In the process of interaction between the sample and the high-frequency evanescent field, the high-frequency information of the sample is transferred to the low-frequency and transmitted to the far field as low-frequency scattered light. In addition, the enhanced E-field can further enhance the scattering signal of the sample. Then, the microscope objective can obtain detailed information about the sample by collecting the scattering light.The imaging resolution and contrast are improved by using the Al patch array as the substrate under DFI, but the resolution is still diffraction limited. To further improve the resolution, samples that cannot be directly resolved by the microscope are observed through a fully immersed SiO2 microsphere under DFI. Sample A placed on sub2 and sub3, as well as sample C on the three substrates are observed. We experimentally find that, with the assistance of SiO2 microsphere, the 300-nm-diameter PS nanoparticle array on sub2 can be clearly resolved, with a magnification of 1.2×, as shown in Fig. 6(a). When the center of the microsphere is well above the edge of the Al patch (the edge is brighter), a few hexagonally arranged Al nanodots at the center of the microsphere, with a center-to-center distance of 125 nm and a nanodot diameter of ∼65 nm, can be vaguely resolved. The magnification is about 2.8×, as shown in Fig. 6(b). The magnification of the metallic array sample is more than twice that of the dielectric array one, due to the excitation of plasmons in metallic nanostructures [7]. The upper right inserts plot the normalized intensity profiles of two adjacent nanoparticles on Figs. 6(a) and 6(b), and the image contrasts are about 0.60 and 0.19, respectively. The other objects remain indistinguishable. According to Fourier optics, when the frequency spectrum of an object is compressed in the frequency domain, the object in the spatial domain will be amplified. Therefore, from the perspective of imaging, the detailed information of the sample is amplified, and from the perspective of the spectrum, the high-frequency spatial spectrum is compressed to be received by the microscope objective.
Fig. 6. Optical images of (a) sample A on sub2, (b) sample C on sub1 under a 6 µm in diameter SiO2 microsphere fully immersed in PDMS. The scale bar is 2 µm.
To investigate the role of the microsphere lens in microsphere-assisted microscopy under DFI, 2D-FDTD is used to simulate the light intensity distribution of a 6 µm SiO2 microsphere fully immersed in PDMS. The refractive indexes of SiO2 and PDMS are 1.46 and 1.4, respectively. Two plane waves (λ=540 nm) are incident obliquely at an angle of 63° from the top. As shown in Fig. 7, the microsphere can focus the incident light, and the light intensity focused on the central imaging region far exceeds that of the incident light [30,31]. When the momentum ${k_{spp}}$ matches the momentum of the focused light and the periodic structure, the SPP modes will be excited, producing the enhanced evanescent field that illuminate the sample. Under the evanescent wave illumination, the largest collectable spatial frequency is $|{{k_{eva}}} |+ 2\pi NA/\lambda $, where ${k_{eva}}$ is the evanescent wave vector and NA is the numerical aperture of the microscope. This means that the microsphere can pick up more high frequency information, compress the high frequency components, and transform it into propagating waves. As a result, the magnified details of the sample can be observed with an ordinary microscope in the form of a virtual image.
Fig. 7. Focusing property of a 6-µm-diameter SiO2 microsphere immersed in PDMS. The wavelength of the incident light is 540 nm and the incident angle is 63°.
4. Conclusion
We demonstrate that surface plasmon-enhanced DFM, which uses a glass slide deposited with a plasmonic Al patch array as the substrate (sub1) to excite the SPPs/LSPRs, can improve the resolution and the contrast in imaging both dielectric objects and metallic ones. A hexagonally closed-packed PS nanoparticle array with a diameter of 300 nm and a hexagonal SiO nanodot one with a nanodot diameter of ∼365 nm and a center-to-center spacing of 450 nm can be clearly resolved. After applying the plasmonic dark-field microsphere-assisted technique, the resolution can be further improved. A hexagonal Al nanodot array with a diameter of ∼65 nm and a center-to-center spacing of 125 nm assembled on sub1 can be just resolved. The enhanced evanescent field illumination and the enhanced E-field on the objects plays an important role in the improvement of imaging resolution and contrast. The surface plasmon-enhanced dark-field microsphere-assisted technique provides a new approach that may have the potential for label-free super-resolution imaging.
Funding
National Natural Science Foundation of China (61475073, 61673287).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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