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This PDF file contains the front matter associated with SPIE Proceedings Volume 9554, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.
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Fluorescence microscopy is an optical microscopy technique which has been extensively used to study specifically- labeled subcellular objects, such as proteins, and their functions. The best possible accuracy with which an object of interest can be localized when imaged using a fluorescence microscope is typically calculated using the Cramer- Rao lower bound (CRLB). The calculation of the CRLB, however, so far relied on an analytical expression for the image of the object. This can pose challenges in practice since it is often difficult to find appropriate analytical models for the images of general objects. Even if an appropriate analytical model is available, the lack of knowledge about the precise values of imaging parameters might also impose difficulties in the calculation of the CRLB. To address these challenges, we have developed an approach that directly uses an experimentally collected image set to calculate the best possible localization accuracy for a general subcellular object in two and three dimensions. In this approach, we fit smoothly connected piecewise polynomials, known as splines, to the experimentally collected image set to provide a continuous model of the object. This continuous model can then be used for the calculation of the best possible localization accuracy.
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Super-resolution imaging has previously been used to identify the position of individual fluorescently-labeled DNA
molecules bound to the surface of gold nanorods. In order to isolate and fit emission from individual fluorophores, a
stochastic photoswitching technique based on shelving the fluorophores into triplet states is used. However, the
reconstructed super-resolution images of the fluorescently-labeled gold nanorods are consistently smaller than the
expected size of the gold nanorod supports. Here, Monte Carlo simulations are used to probe how smaller-than-expected
reconstructed images may be obtained by simultaneous emission events based on short triplet state lifetimes and/or a
high density of fluorescently-labeled DNA on the gold nanorod surface.
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Enhanced Raman scattering from plasmonic nanostructures associated with surface enhanced (SERS) and tip enhanced
(TERS) is seeing a dramatic increase in applications from bioimaging to chemical catalysis. The importance of gapmodes
for high sensitivity indicates plasmon coupling between nanostructures plays an important role. However, the
observed Raman scattering can change with different geometric arrangements of nanoparticles, excitation wavelengths,
and chemical environments; suggesting differences in the local electric field. Our results indicate that molecules
adsorbed to the nanostructures are selectively enhanced in the presence of competing molecules. This selective
enhancement arises from controlled interactions between nanostructures, such as an isolated nanoparticle and a TERS
tip. Complementary experiments suggest that shifts in the vibrational frequency of reporter molecules can be correlated
to the electric field. Here we present a strategy that utilizes the controlled formation of coupled plasmonic structures to
experimentally measure both the magnitude of the electric fields and the observed Raman scattering.
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New Techniques for Nanoimaging and Nanospectroscopy I
In this paper we investigate the expected change in fluorescent decay rate when a fluorophore in aqueous solution is moved to a thin spin-coated layer of poly(vinyl alcohol). We take into account the local field effect due to the change in the refractive index of the medium around the fluorophore and the photonic effect due to the layers. The obtained results are compared with experimental results for the organic dye Atto565 and the fluorescent protein mCherry. We find that the effects for the organic dye can be well described with the model, for the fluorescent protein (FP) the model is less accurate. We discuss the possible explanations for this.
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The unique features of nanocomposite materials depend on the type and size of nanoparticles, as well as their placement
in the composite matrices. Therefore the nanocomposites manufacturing process requires inline control over certain
parameters of nanoparticles such as dispersion and concentration. Keeping track of nanoparticles parameters inside a
matrix is currently a difficult task due to lack of a fast, reliable and cost effective way of measurement that can be used for
large volume samples. For this purpose the Optical Coherence Tomography (OCT) has been used. OCT is an optical
measurement method, which is a non-destructive and non-invasive technique. It is capable of creating tomographic images
of inner structure by gathering depth related backscattered signal from scattering particles. In addition, it can analyse, in a
single shot, area of the centimetre range with resolution up to single micrometres. Still to increase OCT measurement
capabilities we are using additional system extensions such as Spectroscopic OCT (SOCT). With such addition, we are
able to measure depth related parameters such as scattering spectra and intensity of backscattered signal. Those parameters
allow us to quantitatively estimate nanoparticles concentration. Gaining those, information allows to calculate volume
concentration of nanoparticles. In addition, we analyse metallic oxides nanoparticles. To fully characterize nanoparticles
it is necessary to find and differentiate those that are single particles from agglomerated ones. In this contribution we
present our research results on using the LCI based measurement techniques for evaluation of materials with nanoparticles.
The laboratory system and signal processing algorithms are going to be shown in order to express the usefulness of this
method for inline constant monitoring of the nanocomposite material fabrication.
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We performed experimental measurements and theoretical simulation based on an efficient half-space Green’s function
method to investigate the diffraction patterns of light scattering from silicon and ZnO microspheres on a substrate. The
microscopic ellipsometry image for s- and p-polarized reflectance and their phase difference (Rs, Rp, and Δ) was taken
by a modified Optrel MULTISKOP system with rotating compensator configuration for various angles of incidence and
wavelengths ranging from 450nm to 750nm. An 80X objective was used and the pixel size for our image is around
200nm. The images obtained display clear diffraction patterns, which is analyzed by an efficient full-wave simulation
based on half-space Green’s function method. The near-field distributions obtained theoretically are then converted to
far-field images by filtering out the evanescent waves and propagating waves which cannot reach the objective. The
experimental results are then compared with simulated images to gain better understanding of the image patterns. Some
prominent peaks are observed and attributed to resonances related to whispering gallery modes.
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Super-resolution microscopy has revolutionized fluorescence imaging providing access to length scales that are much
below the diffraction limit. The super-resolution methods have the potential for novel discoveries in biology. However,
certain technical limitations must be overcome for this potential to be fulfilled. One of the main challenges is the use of
super-resolution to study dynamic events in living cells. In addition, the ability to extract quantitative information from
the super-resolution images is confounded by the complex photophysics that the fluorescent probes exhibit during the
imaging. Here, we will review recent developments we have been implementing to overcome these challenges and
introduce new steps in automated data acquisition towards high-throughput imaging.
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The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver
endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm)
providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting
higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus
near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is
compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will
discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.
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New Techniques for Nanoimaging and Nanospectroscopy II
Plasmonic coupling of light to free electrons on metallic surfaces allows the confinement of electric fields far below the optical diffraction limit. Scattering processes of molecules placed into these plasmonic ‘hotspots’ are dramatically enhanced[1] which is commonly used to increase the sensitivity of spectroscopic techniques for biological and chemical sensor applications [2, 3]. Strikingly, hardly any measurement technique exists for the direct visualisation and characterisation of the underlying nanoscopic electromagnetic field distributions that either do not perturb the field [3, 4] or require complex electron beam imaging [5]. In this paper we introduce surface enhanced localisation microscopy (SELM), demonstrating the direct visualisation of fields on patterned plasmonic substrates using optical super resolution microscopy [6]. The observed strong photo-blinking behaviour of single molecules in plasmonic fields is exploited in SELM to map electromagnetic field distributions at nanometer resolutions.
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Recently we introduced RESCH microscopy [1] - a scanning microscope that allows slightly refocusing the sample after the acquisition has been performed, solely by performing appropriate data post-processing. The microscope features a double-helix phase-engineered emission point spread function in combination with camera-based detection. Based on the principle of transverse resolution enhancement in Image Scanning Microscopy [2,3], we demonstrate similar resolution improvement in RESCH. Furthermore, we outline a pathway for how the collected 3D sample information can be used to construct sharper optical sections.
[1] A. Jesacher, M. Ritsch-Marte and R. Piestun, accepted for Optica.
[2] C.J.R. Sheppard, “Super-resolution in Confocal imaging,” Optik, 80, 53-54 (1988).
[3] C.B. Müller and J. Enderlein "Image Scanning Microscopy," Phys. Rev. Lett. 104, 198101 (2010).
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Two-photon excitation microscopy (TPEM) provides spatial resolution beyond the optical diffraction limit using the nonlinear response of fluorescent molecules. One of the strong advantages of TPEM is that it can be performed using a laser-scanning microscope without a complicated excitation method or computational post-processing. However, TPEM has not been recognized as a super-resolution microscopy due to the use of near-infrared light as excitation source, which provides lower resolution than visible light. In our research, we aimed for the realization of nonlinear fluorescence response with visible light excitation to perform super-resolution imaging using a laser-scanning microscope. The nonlinear fluorescence response with visible light excitation is achieved by developing a probe which provides stepwise two-photon excitation through photoinduced charge separation. The probe named nitro-bisBODIPY consists of two fluorescent molecules (electron donor: D) and one electron acceptor (A), resulting to the structure of D-A-D. Excited by an incident photon, nitro-bisBODIPY generates a charge-separated pair between one of the fluorescent molecules and the acceptor. Fluorescence emission is obtained only when one more incident photon is used to excite the other fluorescent molecule of the probe in the charge-separated state. This stepwise two-photon excitation by nitro-bisBODIPY was confirmed by detection of the 2nd order nonlinear fluorescence response using a confocal microscope with 488 nm CW excitation. The physical model of the stepwise two-photon excitation was investigated by building the energy diagram of nitro-bisBODIPY. Finally, we obtained the improvement of spatial resolution in fluorescence imaging of HeLa cells using nitro-bisBODIPY.
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The extreme temperature sensitivity of whispering-gallery-mode (WGM) microresonators holds great promise as a detection strategy for single-particle photothermal microscopy and spectroscopy. The detection limit is currently partially constrained by frequency noise from the laser used to probe the cavity resonance wavelength. We present a measurement technique capable of simultaneously detecting backscattered and transmitted light from a wavelengthlocked optical microresonator, with laser intensity noise and frequency noise partitioned into the two independent detection channels. Photothermal mapping of single absorbing nano-objects demonstrates that both methods are capable of high signal/noise, exceeding 30,000:1 in the backscattering channel for a photothermally-induced microresonator resonance shift of 93 fm.
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Tip-enhanced Raman spectroscopy (TERS) can not only provide very high sensitivity but also high spatial resolution, and has found applications in various fields, including surface science, materials, and biology. Most of previous TERS studies were performed in air or in the ultrahigh vacuum. If TERS study can be performed in the electrochemical environment, the electronic properties of the surface can be well controlled so that the interaction of the molecules with the substrate and the configuration of the molecules on the surface can also be well controlled.
However, the EC-TERS is not just a simple combination of electrochemistry with TERS, or the combination of EC-STM with Raman. It is a merge of STM, electrochemistry and Raman spectroscopy, and the mutual interference among these techniques makes the EC-TERS particularly challenge: the light distortion in EC system, the sensitivity, the tip coating to work under EC-STM and retain the TERS activity and cleanliness.
We designed a special spectroelectrochemical cell to eliminate the distortion of the liquid layer to the optical path and obtain TER spectra of reasonably good signal to noise ratio for surface adsorbed molecules under electrochemical potential control. For example, potential dependent TERS signal have been obtained for adsorbed aromatic thiol molecule, and much obvious signal change compared with SERS has been found, manifesting the importance of EC-TERS to reveal the interfacial structure of an electrochemical system.
We further extended EC-TERS to electrochemical redox system, and clear dependence of the species during redox reaction can be identified.
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TERS has emerged over the past decade as a powerful tool for Raman spectroscopy that shows high sensitivity and capability of nano-scale imaging with high spatial resolution. TERS utilizes a metallic nano-tip, which confines and enhances the propagating light into near-field in the close vicinity of the apex. Besides the nano-scale spatial resolution, polarization analysis in TERS is of tremendous advantage, as it allows one to study highly directional intrinsic properties of a sample at the nanoscale. In this study, we have developed a method to analyze the polarization of near-field light in TERS from the scattering pattern produced by the induced dipole in the metallic tip. Under dipole approximation, we measured the image of the dipole at a plane away from the focal plane, where the information about the direction of the dipole oscillation was intact. The direction of the dipole oscillation was determined from the defocused pattern, and then the polarization of near-field light was evaluated from the oscillation direction by calculating the intensity distribution of near-field light We used those evaluated tips to measure nano-images from single-walled carbon nanotubes and confirmed that the contrast of the TERS image depended on the oscillation direction of the dipole, which were also found in excellent agreement with the calculated TERS images, verifying that the polarization of the near-field was quantitatively estimated by our technique.
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We report successful chemically specific high pixel density, high speed (less than 10 minutes per map) TERS imaging of graphene oxide, carbon nanotubes of different chirality, fullerenes and self-assembled layers of organic molecules in both the single-component and complex samples. The spatial resolution routinely obtained in such chemically specific TERS maps is in the 15 - 20 nm range, with the best resolution achieved being 7 nm. The ease of use of the TERS imaging system and high speed TERS imaging capability enabled by advanced hardware and software move TERS closer to become a real life analytical method for chemically specific imaging at the nano scale.
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Surface-enhanced infrared absorption (SEIRA) has been gaining substantial attention by using plasmonic nanoantennas to amplify near-field intensities so that it can extend IR spectroscopy to zeptomolar quantities and ultimately to the sigle-molecule level. Here we report a new nanoantenna for SEIRA detection, consisting of a fan-shaped Au structure positioned at a well-specified distance above a reflective plane with an intervening silica spacer layer. This antenna can be easily tuned to overlap vibrational modes within a broad spectral range from the near-IR into terahertz regimes. Our finite difference time domain (FDTD) simulations reveal a maximum SEIRA enhancement factor of 105 in the antenna junction area, which is corresponding to the experimental detection of 20-200 zeptomoles of octadecanethiol, using a standard commercial FTIR spectrometer. Our optimized antenna exhibits an order of magnitude greater SEIRA sensitivity than previous record-setting designs, which opens new opportunities for using infrared spectroscopy to analyze exceptionally small quantities of molecules.
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Surface-enhanced Raman spectroscopy (SERS) is a powerful technique that yields fingerprint vibrational information with ultra-high sensitivity. However, only roughened Ag, Au and Cu surfaces can generate strong SERS effect. The lack of materials and morphology generality has severely limited the breadth of SERS practical applications on surface science, electrochemistry and catalysis.
Shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) was therefore invented to break the long-standing limitation of SERS. In SHINERS, Au@SiO2 core-shell nanoparticles were rationally designed. The gold core acts as plasmonic antenna and encapsulated by an ultra-thin, uniform and pinhole-free silica shell, can provide high electromagnetic field to enhance the Raman signals of probed molecules. The inert silica shell acts as tunneling barrier prevents the core from interacting with the environment.
SHINERS has already been applied to a number of challenging systems, such as hydrogen and CO on Pt(hkl) and Rh(hkl), which can’t be realized by traditional SERS. Combining with electrochemical methods, we has investigated the adsorption processes of pyridine at the Au(hkl) single crystal/solution interface, and in-situ monitored the surface electro-oxidation at Au(hkl) electrodes. These pioneering studies demonstrate convincingly the ability of SHINERS in exploring correlations between structure and reactivity as well as in monitoring intermediates at the interfaces. SHINERS was also explored from semiconductor surface for industry, to living bacteria for life science, and to pesticide residue detection for food safety. The concept of shell-isolated nanoparticle-enhancement is being applied to other spectroscopies such as infrared absorption, sum frequency generation and fluorescence.
Jian-Feng Li et al., Nature, 2010, 464, 392-395.
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We demonstrate dynamic placement of plasmonic “hotspots” for super-resolution chemical imaging via Surface Enhanced Raman Spectroscopy (SERS). A silver nanohole array surface was coated with biological samples and illuminated with a laser. Due to the large plasmonic field enhancements, blinking behavior of the SERS hotspots was observed and processed using a Stochastic Optical Reconstruction Microscopy (STORM) algorithm enabling localization to within 10 nm. However, illumination of the sample with a single static laser beam (i.e., a slightly defocused Gaussian beam) only produced SERS hotspots in fixed locations on the surface, leaving noticeable gaps in any final image. But, by using a spatial light modulator (SLM), the illumination profile of the beam could be altered, shifting any hotspots across the nanohole array surface in sub-wavelength steps. Therefore, by properly structuring an illuminating light field with the SLM, we show the possibility of positioning plasmonic hotspots over a metallic nanohole surface on-the-fly. Using this and our SERS-STORM imaging technique, we show potential for high-resolution chemical imaging without the noticeable gaps that were present with static laser illumination. Interestingly, even illuminating the surface with randomly shifting SLM phase profiles was sufficient to completely fill in a wide field of view for super-resolution SERS imaging of a single strand of 100-nm thick collagen protein fibrils. Images were then compared to those obtained with a scanning electron microscope (SEM). Additionally, we explored alternative methods of phase shifting other than holographic illumination through the SLM to create localization of hotspots necessary for SERS-STORM imaging.
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Single-particle tracking Live-Cell imaging using Au-NNP(Nanobridged Nanogap Particles) will be presented. Due to a stable plasmonic amplification of Raman signal from the intra-nanogap of Au-NNP, we have a stable and strong Raman signal enough for single particle imaging of Au-NNP in live cell. Multiplex imaging by encoding different Raman dyes into this nanogap and its application will be also discussed.
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Top-down fabrication allows for the realization of morphologically controlled micro/nano devices down to few nanometers scale. We investigate a number of applications based on this fabrication technique, with special emphasis towards nanoplasmonics. In particular, we shall focus on quasi-2D and 3D SERS devices, to move on with TERS structures especially designed to provide superfocusing characteristic. This feature, besides having tremendous impact in improving the TERS spatial resolution, is at the core of a new kind of nanoscopy technique based on hot-electrons transfer.
Finally, we shall provide a flavor of a new paradigm, namely the magnetic field enhancement and concentration from 2D SERS-like nanodevices.
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Earlier, our group proposed a lens made of metallic nanorods, stacked in 3D arrays tapered in a conical shape. This nanolens could theoretically realize super-resolution color imaging in the visible range. The image could be magnified and transferred through metallic nanorods array. Lithography or self-assembly are common ways to fabricate such nanostructured devices. However, to precisely arrange nanorods is challenging due to the limitations to scale down components, and to increase accuracy of assembling particles in large area.
Here we experimentally demonstrated 2D nanolens with long chains of metallic nanorods placed at tapered angles in a fan-like shape to magnify images. In the fabrication, we chemically synthesized gold nanorods coated with CTAB surfactant to ensure a 10 nm gap between the rods for the resonance control of nanolens. And we prepared trenches patterned by FIB lithography on a PMMA coated glass substrate. The different hydrophobicity of PMMA and CTAB coats enabled to optimize capillary force in gold nanorod solution and selectively assemble nanorods into hydrophilic trenches. Finally, we obtained 2D nanolens after lift-off of the PMMA layer.
We numerically estimated the resonance property of nanorods chain and found a broad peak in the visible range located at a wavelength of 727 nm. The broadness of this peak (~178 nm) confirms that a broad range of wavelength can be resonant with this structure. This phenomenon was also confirmed experimentally by optical measurements. These results show that the combination of lithography and self-assembly has the potential to realize plasmonic nanolens.
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Graphene has high potential for becoming the next generation material for electronics, photonics and optoelectronics. However, spatially controlled modification of graphene is required for applications. Here, we report patterning and controlled tuning of electrical and optical properties of graphene by laser induced non-linear oxidation. We use four wave mixing (FWM) as a key method for imaging graphene and graphene oxide patterns with high sensitivity. FWM produces strong signal in monolayer graphene and the signal is highly sensitive to oxidation providing good contrast between patterned and non-patterned areas. We have also performed photo-oxidation and FWM imaging for air suspended carbon nanotubes.
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We report multiplexed spectral imaging of plasmon resonance shifts in a simple setup, capable of resolving single protein binding events with a high signal to noise ratio of up to 10 for single Fibronectin (450 kDa) proteins. We directly record 2-dimensional, spectrally dispersed images of multiple gold nanorods. The spectra are corrected via an overlapped particle image that correlates arbitrary particle positions to the reference slit position, allowing us to use the full 2D space of the sensor. Together with the simple experimental implementation, our approach paves the way towards routine and efficient label-free detection down to the single molecule level.
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Single-molecule spectroscopy (SMS) at low temperature was used to study the spectral properties, heterogeneities and spectral dynamics of the chlorophyll a (Chl a) molecules responsible for the fluorescence emission of photosystem I (PS I). The fluorescence spectra of single PS I complexes are dominated by several red-shifted Chl a molecules categorized into red pools called C708 and C719. By polarization dependent measurements we demonstrate spectrally separate emissions corresponding to C708 and C719 in single PS I monomers and trimers. Moreover, we compared the results of SMS polarization dependent between monomeric and trimeric PS I complexes and give an estimation for the orientation between these red pools. As a consequence, we get new insight into the energy transfer towards and between the red Chl a molecules in PS I complexes.
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