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Plasmonic Gratings


Figure 5 : Single-molecule images of Cy5 molecules from the sample imaged in Figure 3c showing the size and separation of nanostructures. (a) Representative fl uorescence image of one time-frame fl uorescence raw image before super-resolution analysis. (b) Super-resolution image after localization microscopy analysis (zoomed in detail). (c) Corresponding 3D intensity map of nanoprotrusions. (d) Corresponding time trace to resolve nanostructures at different positions. (e) Intensity vs. distance plot to show the size obtained from super-resolution fl uorescence image. (f) Height vs. distance plot of AFM image to show the size of nanoprotrusions. Insets are corresponding fl uorescence and AFM images showing cutting curves for profi le analysis and FWHM for each fi tted peak. Reproduced with permission from [ 19 ] with permission, copyright 2016 RSC.


image had been signifi cantly improved. The minimum separation distance was reduced to ~65 nm, and the full width at half maximum (FWHM) of the emission was reduced to ~40 nm ( Figure 5e ). This separation distance and FWHM correlate well with separation and size of nanoprotrusions measured with AFM at several locations ( Figure 5f ). Due to the presence of a conformal PMSSQ coating with embedded dye molecules, we could not obtain surface information with AFM aſt er fl uorescence imaging, but we are currently developing methods to track specific molecules in both fl uorescence and AFM.


Discussion


We have developed a plasmonic grating platform that can simply and eff ectively extend the resolution limits of any fl uorescence microscope by behaving as a far-fi eld superlens (FSL). An FSL operates by enhancing the evanescent high spatial frequency information at the surface and converting it into propagating information that is detectable in the far-fi eld. Super-resolution imaging using a metallic grating as an FSL has also been accomplished by other research groups but with brightfield imaging rather than fluorescence imaging [ 12 , 35 , 36 ]. Super-resolution sample information under bright-fi eld conditions can be obtained using silver windows to block the 0-order diffracted light containing diffraction-limited


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information and collecting the ±1-order diff raction containing the super-resolution information [ 7 ]. However, in fl uorescence imaging, SPCE is used to obtain super-resolution information. T e SPCE eff ect occurs when an excited fl uorophore within 100 nm from the grating surface non-radiatively transfers energy back to the plasmonic grating as a radiative surface plasmon, which is then emitted as a photon at the SPR coupling angle [ 37 ]. In the case of fl uorescence imaging, the excitation background light refl ected from the sample is cut off by the fluorescence filter cube, but fluorescence emitted via SPCE at the ±1 diff raction order is free to pass through the fi lter cube. The reflected excitation light can be further reduced using an excitation polarizer with a randomly polarized light source, such as a halogen or xenon arc lamp. Based on the SPR dispersion relation, p-polarized light is the only polarization that can excite oscillations at the grating/dielectric interface, also known as surface plasmon polaritons. T e s-polarized light is inherently unable to generate surface plasmons because the electric component of the EM wave is parallel to the grating and dielectric interface. With use of an excitation polarizer, the s-polarized light that is typically refl ected from the sample can be eliminated before reaching the sample.


Ultimately, the GLAD grating platform more specifi cally extends the resolution limits: (1) it converts incident light into intense electric fi elds at the surface that can be further concentrated at nanoprotrusions and nanogaps to create


www.microscopy-today.com • 2017 January


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