Nanospeckle Illumination Microscopy (NanoSIM) utilizes plasmonic nanoisland structures to enable super-resolution surface imaging of live cells. By analyzing the intensity fluctuations of plasmonic nanospeckles, we achieved three-fold improved spatial resolution and the ability to identify multiple cellular structures. Experimental results demonstrate the potential of NanoSIM as an effective and versatile tool for investigating dynamic cellular processes within live cell membranes of HeLa cells, providing crucial insights into complex cellular interactions.
Various plasmonic nanostructure-based substrates are used to detect biological signals beyond the diffraction limit with a high signal-to-noise ratio. These approaches take advantage of excitation of localized surface plasmon to acquire high-frequency biological signals while preserving photon energy. Numerous techniques, including focused ion beam, electronbeam lithography, and reactive ion etching, have been used to fabricate plasmonic substrates. However, these fabrication techniques are time and resource-consuming. In contrast, disordered nanostructure-based substrates have attracted interests due to the easy fabrication steps and potential cost savings. Metallic nanoisland substrates, for instance, can be mass-produced using thin film deposition and annealing without lithographic process. In this work, we have investigated nanospeckle illumination microscopy (NanoSIM) using disordered near-field speckle illumination generated by nanoisland substrate. Selectively activated fluorescence wide-field images were obtained by nanospeckle illumination generated on the nanoisland substrate. Super-resolved fluorescence images were reconstructed by an optimization algorithm based on blind structured illumination microscopy. Experimental studies of various biological targets including HeLa cell membranes were performed to demonstrate the performance of NanoSIM. Using NanoSIM, we were able to improve spatial resolution of ganglioside distribution in HeLa cells targeted by CT-B by more than threefold compared to the diffraction-limited images. Note that the accessibility of super-resolution imaging techniques can be enhanced by the nanospeckle illumination of disordered metallic nanoislands. These results may be used in imaging and sensing systems that work with detecting biological signals beyond diffraction limits in various applications.
We have investigated the feasibility of disordered plasmonic nanocomposites for super-resolution imaging. Annealing-based nanocomposite substrate has a great potential in biomedical optical and sensing technology because it can be mass-produced without difficult manufacturing processes. We introduce a new approach for wide-field super-resolution fluorescence imaging based on the nanocomposite island substrates, which we call nanospeckle illumination microscopy (NanoSIM). Near-field speckle patterns produced on disordered nanoisland substrates can help reconstruction of high-resolution fluorescence images with appropriate basis images. We have acquired basis images using azimuthal scanning illumination (ASI). Each ASI produces nonuniform nanoscale near-field speckles which can excite fluorescent dyes within a subdiffraction-limited area. While exploiting the random nature of plasmonic nanocomposite, NanoSIM does not require any specific polarization state to be maintained for ASI. We have tested NanoSIM to obtain super-resolved mages of molecules on the HeLa cell membrane. The full-width-at-half maximum was shown to improve by more than three times over the diffraction-limit with 360 basis images. Reconstructed images of gangliosides distribution on the HeLa cell suggest that fewer basis images may produce almost the same resolution with a shorter computation time. The optical resolution and sensitivity of disordered plasmonic substrate can be further enhanced by controlling the geometrical features of nanoislands structure.
Beyond structured illumination microscopy (SIM) which uses diffraction-limited light illumination, specially designed nanostructures such as metallic nanoantenna arrays generating localized surface plasmon have been developed to expand the frequency information without increasing photon energy. In this study, disordered temperature-annealed nanocomposite islands were used to create random distribution of nanospeckles because nanoisland substrates can be mass-produced in a large observation area by thin film deposition and annealing process. In our nanospeckle illumination microscopy (NanoSIM) system, azimuthal scanning illumination (ASI) on nanoislands creates a randomly localized nearfield distribution that induces an arbitrary number of fluorescence images. By the difficulty of obtaining structured illumination patterns of random nanostructures, images were reconstructed using a modified blind-SIM algorithm which fits well with the ASI system. A 100 nm fluorescent nanobead experiment confirms that NanoSIM provides resolution enhancement of spatial information in good agreement with the results obtained from AFM images. We emphasize that using random nanospeckles of disordered nanocomposite islands can provide highly accessible super-resolution. The results can be applied to imaging and sensing techniques, such as switching-based multi-channel microscopy.
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