Dark-field microscopy (DFM) is a widely used imaging tool due to its high-contrast capability in imaging label-free specimens. However, traditional DFM requires optical alignment to block the oblique illumination and the resolution is diffraction-limited to wavelength scale. In this work, we present a single frame super resolution method using plasmonic dark-field microscopy (PDF) and deep learning image reconstruction algorithm. Based on our framework, we demonstrated more than 2.5 times resolution enhancement on various objects. We highlight the potential of our technique as a compact alternative of traditional DFM with enhanced spatial resolution.
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.
Surface plasmon, collective electron oscillation induced by light absorption in noble metals, has received renewed attention that opens a new area of photonics research in what is known as thermoplasmonics. As thermoplasmonics develops, opto-thermal response measurement of a single nanostructure becomes essential. In this study, we propose a collection-type near-field scanning optical microscopy (NSOM) that can simultaneously measure light absorption and near-field enhancement on a single nanostructure. We analyzed light absorption from optically induced thermal expansion while measuring a near-field coupled with the NSOM tip. We have observed discrepancy and nonlinearity of angular spectrum between light absorption and near-field enhancement on gold thin films and compared with simulation results based on iterative opto-thermal analysis. We were able to determine the cause of the axial shift on the NSOM and the mechanisms by which the discrepancy may ariss. The proposed technique can also acquire optical characteristics of a single disk in a periodic array of gold nanodisks, and even measure the gaps between the disks. Furthermore, we expect the proposed technology to be extended to measuring near-field thermal characteristics of more complicated structure such as metamaterials.
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 investigate the method to analyze interferometric plasmonic microscopy (IPM) images using a deep learning approach. An IPM image was generated by employing an optical model: the image intensity was formed by reflected and scattered fields. Convolutional neural network was utilized for the classification of IPM images. Conventional detection method based on fourier filtering was taken for comparison with the proposed method. It was confirmed that deep learning improves the performance significantly, in particular, robustness to noise. These results suggested applicability of deep learning beyond IPM images with higher efficiency.
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.
We have investigated the plasmonic effect of a gold thin film on the optical properties under a range of combinations of incident wavelengths, incident angles and polarization states, while assuming various film thicknesses. Theoretical calculation was performed with rigorous coupled-wave analysis based on the temperature-dependent Drude-Lorentz dispersion model. The calculation method considers the effects of absorption, which is converted to heat in a gold thin film and can affect material parameters such as permittivity. Experimentally, light absorption and field enhancement factor were directly measured using near-field scanning optical microscopy. We have also measured the near-field distribution and thermal effects in the gold thin film. Absorption and field enhancement experimentally measured using three incident wavelengths of 488, 532, and 721 nm for a thin gold film with thicknesses 20, 50, and 70 nm showed good agreement with calculated data. Also observed was the disparity between the incident angles that correspond to maximum absorption and highest field enhancement. The results can help understand the thermal effects on optical properties of plasmonic nanostructures for applications in biological imaging and sensing techniques.
We demonstrated gold nanodimer arrays could improve the signal-to-noise ratio (SNR) of fluorescence correlation spectroscopy (FCS). In this research, we explore the feasibility of plasmon-enhanced FCS for biomolecular study using a nanodimer array whose gap size was 18 nm. Fluorescence nanobead with a diameter of 40 nm was first examined to verify if gold nanodimer arrays can enhance SNR of fluorescence and scattering intensities. We emphasize that plasmon-enhanced FCS can improve the precision for analyzing the dynamics of the particle by combining scattering characteristics of nanodimer arrays to surface plasmon resonance imaging technique. We have also observed the fluorescence enhancement and plasmon scattering in the movement of lysosome in HEK293 cells. It was found that we could measure diffusion properties such as diffusion coefficients and anomalous exponents with a low standard deviation.
In this study, we have used scanning probe microscopy (SPM) to validate temperature-dependent thermo-plasmonic calculation which is called iterative opto thermal analysis (IOTA). We have applied temperature-dependent Drude-Lorentz model to IOTA. To solve wave-coupled heat transfer equation, finite element method based multi-physics analysis tool has employed with wave optics module and heat transfer module under the proper boundary and initial conditions. For this study, various plasmonic structures were considered to acquire temperature and plasmonic field enhancement using IOTA and SPM experiments. As a result, we have improved plasmonic analysis with consideration of temperature-dependence.
We investigate a way to detect images of surface plasmon scattering using deep learning approach. Unlike fluorescence imaging, the image of surface plasmon scattering shows much worse resolution due to propagation length of surface plasmon polariton. In this work, deep learning approach is taken to address this issue and to discriminate multiple target objects under complex and noisy environment. Conventional detection method based on fourier filtering and deconvolution was employed to compare the performance of the proposed method. It was shown that deep learning improves the accuracy by about six times, and especially more useful in noisy environment.
We have investigated the excitation of fluoresce molecules using nanoscale light confinement on the plasmonic nanostructures. We have fabricated gold nano-dimer arrays whose diameter and height were 100 and 20 nm respectively on 20-nm gold film with BK7 substrate with a period of 746 nm. We have calculated the field distribution by three dimensional finite-difference time-domain (FDTD) method and confirmed the field localization on the dimer’s gap whose size was 18 nm. The field confinement was induced by a light source at 671 nm and experimentally measured by near-field scanning optical microscopy (NSOM) under same incident condition given at FDTD calculation. The angle of surface plasmon resonance (SPR) was chosen to enable both fluorescence microscopy and SPR microscopy simultaneously. Given a resonance angle, a dimer could provide a subdiffraction-limited observation volume to study the dynamics of fluorescence molecules and a highly sensitive light scattering probe as a nanoantenna. By employing dual microscopic images, we could separate the fluorescence excitations within a subdiffracion-limited volume from those outside the volume. We calculated scattering intensities of fluorescence nanobeads on the dimer nanoantenna to assure the presence within an observation volume when they diffuse near the dimer nanoantenna with Brownian motion. We have applied the subdiffraction-limited volume to fluorescence correlation spectroscopy (FCS) to measure the dynamics of fluorescence molecules with high signal-to-noise ratio, with additional spatial analysis from SPR microscopy. The result of imaging FCS using gold nano-dimer with SPR microscopy shows novel applications for nanoscale sensing and imaging methods.
KEYWORDS: Near field, Gold, Switching, Nanostructures, Plasmonics, Near field scanning optical microscopy, Atomic force microscopy, Quantitative analysis
We investigate switching near-field distribution on metal random nanoislands by changing the direction and the angle of light incidence in 14 channel modes. Distribution of the near-fields induced by different channel modes was calculated by finite difference time domain method. The size of near-fields under oblique channel modes ranges 48 - 77 nm in contrast to 127 - 145 nm with normal incidence. Quantitative analysis of near-field position was performed relative to nanoislands. Near-field position was largely well aligned with the direction of incident channel modes. Switching near fields was experimentally confirmed in two ways, first by measurement of fluorescence intensity and by NSOM. Fluorescence experiment was conducted by using bare glass substrate and gold nanoislands in seven channel modes. Fluorescence intensity on bare glass substrate shows symmetric intensity changes with channel modes. However, fluorescence intensity on gold nanoislands was found to be asymmetric. For quantitative analysis, mean-squared error (MSE) was calculated by defining fluorescence intensity as a 7D vector. Distribution of MSE in case of gold nanoislands was broader than on bare glass substrate. In other words, near fields induced on gold nanoislands were switched more strongly than bare glass substrate. Also, near fields induced on nanoislands were measured directly using NSOM in two channel modes. It was confirmed that spatial positions of near-fields depend on channel modes. The results of this study suggest that the near fields can be controlled by adjusting channel modes, which opens possibilities of highly sensitive and super-resolved detection and imaging.
It is required to reduce the excitation volume of fluorescence with enhanced field intensity to apply fluorescence correlation spectroscopy (FCS) technique to the investigation of biological molecules. Field localization induced by plasmonic nanostructures enables measurements of molecular dynamics under high concentration exhibiting high signal-to-noise (SNR) ratio. To achieve this goal, we have investigated the feasibility of plasmonic monomer and dimer nanostructures for FCS techniques. We have studied field enhancement and localization induced by different gold monomer arrays whose shapes were circle, rhombus and triangle and also gold dimer arrays which had a gap of 10 nm. These plasmnoic nanostructures were considered to be on the gold film and glass substrate with chrome adhesion layer. We could shift the peak wavelength of field enhancement by changing the dimensions of nanostructures to spectrally overlap the field enhancement to excitation and emission spectrum of fluorophores. In the case of dimer configuration having a 90-nm diameter and a 20-nm height, we have induced the near-field localization with a light source at 671 nm whose dimension was 18×6×6 nm^3 with an enhanced field intensity by 500 times in comparison with a incident light. The field distribution was analyzed numerically and experimentally using finite-difference-time-domain method and near-field scanning optical microscope. We could measure the diffusion coefficients of 50-nm fluorescent beads with improved SNR which was found to be 44.6×10-14 m^2/s. Results would include the diffusion mapping of fluorescence molecules using imaging FCS technique to show the plasmnoic nanostructures is applicable to nanoscale FCS for study of molecular biology.
KEYWORDS: Nanolithography, Plasmonics, Near field optics, Near field, Biosensors, Target detection, Lithography, Biosensing, Nanostructures, Thin films
The detection sensitivity of surface plasmon resonance (SPR) biosensors has been improved by employing colocalization of spatial distribution of electromagnetic near-fields and detection molecules. We have used plasmon nanolithography to achieve light-matter colocalization on triangular nanoaperture arrays and optimized array configurations to improve colocalization efficiency. Streptavidin-biotin interactions were measured to validate the concept. It was confirmed that colocalized distributions of target binding and localized near-fields produced larger optical detection sensitivity. The colocalized detection was also shown to come with wider dynamic range than noncolocalized detection. The effective limit-of-detection of colocalized measurements was on the order of 30 pM. The colocalized detection sensitivity was estimated to be below 1 fg/mm2 in a 100-nm deep evanescent area, an enhancement by more than three orders of magnitude over conventional SPR sensor.
KEYWORDS: Near field, Super resolution, Light sources, Photoacoustic microscopy, Metals, Plasmonics, 3D photoacoustic microscopy, Near field scanning optical microscopy, Biomedical optics, Luminescence
Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to ~200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.
Plasmonic nanostructures enable field confinement which is locally amplified within sub-diffraction limited volume. The localized near-field can be useful in many biomedical sensing and imaging applications. In this research, we present the near-field characteristics localized by plasmonic nano-post arrays for biomedical spectroscopy. Circular gold nano-post arrays were modeled on gold and chrome films fabricated on a glass substrate whose thickness was 50, 20 and 2 nm, respectively. The nano-post arrays were fabricated with an e-beam lithography and a diameter of the post was 250 nm with periods varied as 500, 700, and 900 nm. The field localization produced by nano-posts was induced by angled illumination with a total internal reflection fluorescence microscope objective lens and measured by a near-field scanning optical microscope (NSOM). The NSOM has a tapered fiber probe with a 70-nm aperture and was a continuous-wave laser whose wavelength is 532 nm as light source. Incident TM-polarized light exhibited field localization on one side of an individual gold nano-post. When the direction of light incidence was changed opposite, localized field was switched to the opposite edge of the circular nano-post. We performed 3D finite difference time domain s for the field calculation and confirmed the localized field distribution at given illumination angles. We also discuss the potential applications of plasmonic field localization for analysis of biomolecules, cells, and tissues.
Locally amplified near-fields can be induced with nanostructures within a sub-diffraction-limited volume, which is useful for biomedical imaging and sensing applications. Employment of field localization in the biomedical applications where the pulsed light is used necessitates the spatial and temporal characteristics of fields near nanostructures. We considered the gold nano-post arrays of three different shapes to localize the near-fields which are circular, rhombic, and triangular. They were modeled to be located on an ITO film and a quartz substrate with periods changing from 300 to 900 nm by 200 nm. Their size changes from 50 to 250 nm which corresponds to the radius for the case of circular nanoposts and the distance between the center and the vertices for equilateral rhombic and triangular nanoposts. Numerical calculation of near-fields at the top of nanoposts was performed with finite difference time domain method when the Gaussian pulses at center wavelengths of 532, 633, and 850 nm were normally incident. Near-fields localization occurred mainly at vertices of the nanoposts, which makes the triangular nanoposts of primary interest with an observation of the strongest field intensity within a diffraction limited field-of-view. The observed fields on the triangular vertices were enhanced by 7.85, 51.54, and 7268 when the center wavelengths were 532, 633, and 850 nm respectively. Their temporal peaks were delayed by 2.05, 4.03, and 14.49 fs, which indicates the correlation between field enhancement and time delay associated with electron damping process. It was shown that with rhombic and triangular nanoposts fields can be localized below 10 nm on vertices and their signal-to-noise ratio increased with a larger period.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.