doi:10.1017/S1551929516001103 42 www.microscopy-today.com • 2017 January Super-Resolution Light Microscopy Using Plasmonic Gratings Aaron Wood, 1 Biyan Chen, 2 Joseph Mathai, 2 Sangho Bok, 2 Sheila Grant, 1 Keshab Gangopadhyay, 2 Peter V. Cornish, 3 and Shubhra Gangopadhyay 2 * 1 Department of Bioengineering, 139 and 141A Engineering Building West, University of Missouri, Columbia, MO 65211 2 Department of Electrical and Computer Engineering, 139 and 141A Engineering Building West, University of Missouri, Columbia, MO 65211 3 Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211 *gangopadhyays@missouri.edu Abstract: A novel platform for super-resolution imaging has been devised that employs plasmonic gratings fabricated using glancing angle deposition (GLAD) of silver. GLAD was found to produce a large population of unique nanostructures over the entire plasmonic grating. These nanostructures excite nearby fluorescent molecules to improve spatial resolution to sub-diffraction limit distances while also increasing signal-to-noise ratio (SNR). For example, the improved localization precision produces 65 nm image resolution on a highly concentrated fluorescent sample. These inexpensive plasmonic GLAD gratings have potential to improve fluorescent intensity and resolution over a wide range of applications. Introduction Super-resolution microscopy, where the resolution can break the diffraction limit, plays a critical role in advanced biological research. Super-resolution microscopy enables scientists to observe the movement and interaction of individual molecules and nanoscale features [1–6]. Several super-resolution techniques have been developed over the past few years that have led to a better understanding of biological systems. Tese include near-feld scanning optical microscopy (NSOM) [7–12], far-feld superlens [13], photo-activated localization microscopy (PALM) [14, 15], and stochastic optical reconstruction microscopy (STORM) [16, 17]. Tese techniques typically use mathematical models and a series of difraction-limited images or complicated optical, mechanical, and electrical setups to increase the spatial resolution to sub-difraction limit levels. However, there are still limitations to certain super- resolution techniques. For example, tip-based techniques (such as NSOM) require lengthy scan times when imaging large sample areas because the tip must be rastered over the entire surface [12, 18]. Unlike tip/probe-based techniques, localization microscopy (PALM and STORM) pinpoints the centroids of well-separated single molecules using hundreds or thousands of images to reconstruct a few super-resolution images. Te location precision can be greatly increased by combining this technique with microscopes equipped with optical sectioning capabil- ities, such as total internal refection fuorescence microscopy (TIRFM), which enhances the signal-to-noise ratio (SNR) [3]. In this article we describe a novel fuorescence enhancement platform, recently published in Nanoscale [19], that further enhances the SNR without the need for complex optical setups. This platform uses a plasmonic grating with unique nanostructures grown on the surface. Plasmonic gratings, that is, ridge-groove structures of a noble metal such as gold or silver, are able to generate intense electromagnetic fields (EM felds) when light at specifc wavelengths shines onto the grating surface at specifc angles. Tis phenomenon is known as surface plasmon resonance (SPR). Afer growing additional nanostructures on the grating, we found that these nanostruc- tures can concentrate the EM feld generated by the plasmonic grating to greatly enhance the number of photons emitted by nearby fuorescent molecules. When compared with STORM/ PALM, the enhanced SNR of the plasmonic grating enables single-molecule localization precision over a wide range of fluorophore concentrations using a simple epifluorescence microscope, eliminating the need for TIRFM. Additionally, plasmonic gratings have an inherently wider coupling angle range than prism-based plasmonics, which eliminates the need for laser excitation with fine-angle control and allows users to excite the grating with the illumination from a typical microscope objective. Materials and Methods Grating microstructure. Plasmonic gratings are relatively inexpensive to produce compared to those produced by electron- beam lithography and may be fabricated from the grating structures found on high-defnition DVDs or Blu-ray discs. Afer producing the grating profle in polymethylsilsesquioxane (PMSSQ) on a microscope slide using microcontact lithog- raphy, the gratings are coated with a thin flm of silver metal. Te latter process is accomplished via GLAD where the silver is deposited at an angle relative to the grating surface [20–25]. In previous publications, we developed normally deposited silver plasmonic gratings with random nanogaps to enhance single molecule fuorescence [20] and to detect DNA oscillation [21]. While a conformally deposited silver flm provides only some of the plasmonic properties needed, the GLAD process forms a silver flm with an abundance of diferent nanostructures such as nanoprotrusions and nanogaps, as seen in Figures 1a and 1b [19]. Nanoprotrusions are protruding silver grains forming on top of the grating ridges that can generate “hotspots” via localized surface plasmon resonance (LSPR) that excites nearby fuorophores. Nanogaps are narrow crevices that form at the base of the grating ridge in the shadowed region where the fux of silver was greatly reduced. During the development of these GLAD gratings, it was found that a deposition angle with respect to the surface of α = 60° produced the highest density of these two nanostructures. Tis is advantageous because a high density of plasmonic hotspots enables instantaneous imaging over a large area instead of the point-by-point scanning used in NSOM and provides a better SNR (~28) than more complicated and expensive systems (NSOM SNR ~7) [26] and much higher than the minimum SNR for single molecule imaging (~3) [27]. Downloaded from https://www.cambridge.org/core. 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