Localized surface plasmon was formed with plasmonic nanohole arrays for measurement of the Raman spectrum in polystyrene beads. For the selective acquisition of enhanced Raman signals, we used the surface-enhanced Raman spectroscopy (SERS) system employing particle manipulation within the electrode chip. This integration involves the amalgamation of metal nanostructures and microelectrodes to assess the electrophysiological characteristics of microparticles. The manipulation of particle movement, contingent upon their electrical properties such as conductivity and permittivity, can be controlled through modulation of the frequency of the applied alternating current field. In our integrated system, Raman spectroscopy can be conducted with enhanced intensity. Real-time measurement of enhanced Raman scattering is achieved by controlling the direction of force exerted on single particle and selectively trapping it on the nanostructured surfaces.
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.
KEYWORDS: Light sources and illumination, Speckle, Microscopy, Reflection, 3D image processing, Super resolution, Biological imaging, 3D image reconstruction
Optical super-resolution microscopy has revolutionized imaging in the lateral axis, enabling nanoscale structure visualization with unprecedented detail. However, achieving high axial resolution along the z-axis remains challenging. In this study, nanoscale fluctuation-enhanced axial localization microscopy addresses this issue by incorporating metallic structures, specifically reflective optical devices, into dynamic speckle illumination microscopy. By controlling light waves within the fluidic chip, these metallic devices enable super-resolution to be achieved not only in the lateral direction but also along the z-axis, all in a cost-effective manner. Experimental investigation using 100-nm fluorescent beads and the U-87 MG cell membrane demonstrates axial-resolving performance of fluctuation-enhanced imaging compared to conventional methods. The application of an optical fluctuation-based reconstruction algorithm further allows the extraction of 4-fold enhanced axial information over diffraction-limited system.
When a microparticle is exposed to an alternating current (AC) electric field, the polarized particle is moved by the interaction between the dipoles and the AC electric field under dielectrophoresis (DEP). The DEP force is widely used for manipulation of microparticles in diverse practical applications such as 3D manipulation, sorting, transfer, and separation of various particles such as living cells. In this study, we propose integration of surface-enhanced Raman spectroscopy (SERS), an extremely sensitive and versatile technique based on Raman scattering of molecules supported by nanostructured materials, with DEP using a microfluidic device, i.e., the microfluidic device combines metal nanostructures and microelectrodes to characterize electrophysiological and biochemical properties of biological cells. The movement of particles, which varies depending on the electrical properties such as conductivity and permittivity of particles, can be manipulated by the cross-frequency change, which is one of the DEP properties. Raman spectroscopy using this DEP-SERS integrated system can be performed with an improved signal-to-noise ratio by determining the direction of the DEP force applied to the cells with respect to the applied AC power and collecting them on the nanostructure.
Metallic nanostructures have the potential to be used in a variety of applications related to sensing and imaging biological molecules due to their ability to enhance the way molecules absorb and emit light. However, the interaction between metallic nanostructure and molecules can give rise to difficulty with determining precise molecular positions and orientations and therefore pose major challenges in the field of super-resolution imaging. In this work, we used axially defocused imaging to analyze the interaction between a single fluorescent molecule and a metallic nanostructure. In addition, a pattern matching algorithm was used to analyze the images, explore the interaction between the molecule and the nanostructure and thereby determine the lateral position. The accuracy was found to improve while the degree was dependent on the dipolar orientation and the distance between dipole and nanostructure. This approach has the potential to improve the reliability of using metallic nanostructures for imaging and sensing in the future and opens up new possibilities for various imaging and sensing methods.
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.
This work describes extreme light localization for intracellular molecular imaging and sensing with a high signal-to-noise ratio and precision. We explore localization techniques by which achievable resolution may be customized for subcellular dynamics of molecular complexes. We have also conducted plasmon-enhanced fluorescence correlation spectroscopy of cellular organelles with improved precision. The approach was extended to switching-based light localization to circumvent the diffraction limit and to use random disordered composite metallic islands for improved structured light microscopy. Extreme light localization also proves useful for enhancing Raman microscopy. Localization-based super-resolved Raman microscopy and techniques in combination with structured illumination will be discussed.
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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