Surface Plasmon Microscopy Research Articles

High-resolution simultaneous microscopy of refractive index and fluorescent intensity distributions by using localized surface plasmons

We propose a localized surface plasmon microscope that provides simultaneous imaging of refractive index and fluorescent intensity distributions. We show experimental images of fluorescent and transparent particles under circular pupil illumination to confirm simultaneous high-resolution imaging. Furthermore, we investigate applicability of annular pupil illumination employing two axicons to improve energy efficiency in the fluorescent imaging and find that a brighter image is obtainable by maintaining high spatial resolution for both imaging modes.

Quantitative plasmonic measurements using embedded phase stepping confocal interferometry

In previous publications [Opt. Express 20, 7388 (2012), Opt. Express 20, 28039 (2012)] we showed how a confocal configuration can form an surface plasmon microscope involving interference between a path involving the generation of surface plasmons and one involving a directly reflected beam. The relative phase of these contributions changes with axial scan position allowing the phase velocity of the surface plasmon to be measured. In this paper we extend the interferometer concept to produce an 'embedded' phase shifting interferometer, where we can control the phase between the reference and surface plasmon beams with a spatial light modulator. We demonstrate that this approach facilitates extraction of the amplitude and phase of the surface plasmon to measure of the phase velocity and the attenuation of the surface plasmons with greatly improved signal to noise compared to previous measurement approaches. We also show that reliable results are obtained over smaller axial scan ranges giving potentially superior lateral resolution.

Wavelet-based decomposition of high resolution surface plasmon microscopy V (Z) curves at visible and near infrared wavelengths

Surface plasmon resonance is conventionally conducted in the visible range and, during the past decades, it has proved its efficiency in probing molecular scale interactions. Here we elaborate on the first implementation of a high resolution surface plasmon microscope that operates at near infrared (IR) wavelength for the specific purpose of living matter imaging. We analyze the characteristic angular and spatial frequencies of plasmon resonance in visible and near IR lights and how these combined quantities contribute to the V(Z) response of a scanning surface plasmon microscope (SSPM). Using a space-frequency wavelet decomposition, we show that the V(Z) response of the SSPM for red (632.8 nm) and near IR (1550 nm) lights includes the frequential response of plasmon resonance together with additional parasitic frequencies induced by the objective pupil. Because the objective lens pupil profile is often unknown, this space-frequency decomposition turns out to be very useful to decipher the characteristic frequencies of the experimental V(Z) curves. Comparing the visible and near IR light responses of the SSPM, we show that our objective lens, primarily designed for visible light microscopy, is still operating very efficiently in near IR light. Actually, despite their loss in resolution, the SSPM images obtained with near IR light remain contrasted for a wider range of defocus values from negative to positive Z values. We illustrate our theoretical modeling with a preliminary experimental application to blood cell imaging.

Surface plasmon microscopic sensing with beam profile modulation

Surface Plasmon microscopy enables measurement of local refractive index on a far finer scale than prism based systems. An interferometric or confocal system gives the so-called V(z) curve when the sample is scanned axially, which gives a measure of the surface plasmon propagation velocity. We show how a phase spatial light modulator (i) performs the necessary pupil function apodization (ii) imposes an angular varying phase shift that effectively changes sample defocus without any mechanical movement and (iii) changes the relative phase of the surface plasmon and reference beam to provide signal enhancement not possible with previous configurations.

Localized surface plasmon microscopy of submicron domain structures of mixed lipid bilayers.

We propose scanning localized surface plasmon microscopy of mixed lipid bilayers with submicron domain structures. Our observation technique, which employs localized surface plasmons excited on a flat metal surface as a sensing probe, provides non-label and non-contact imaging with the spatial resolution of ∼ 170 nm. We experimentally show that submicron domain structures of mixed lipid bilayers can be observed. A detailed analysis finds that the domains are classified into two groups.

Uncovering phase maps from surface plasmon resonance images: Towards a sub-wavelength resolution

Abstract We present an original method for uncovering phase maps from surface plasmon resonance microscopy images. The phase images obtained from the recording of V ( Z ) maps with a scanning surface plasmon microscope are affected by zero-mean uncorrelated noise and mechanical drifts. We propose to replace standard phase unwrapping methods by the computation of the local derivative of the phase Δ ϕ / Δ Z from the V ( Z ) complex field. Phase unwrapping basically relies on a smoothness constraint of the phase field, which is severely hampered by the noise. Applications of the proposed derivation to interferometric plasmon phase images demonstrate a superior ability of restoring phase maps, preserving their discontinuities, together with an effective noise smoothing performance, irrespective of locally varying coherence characteristics.

Surface plasmon microscopy: resolution, sensitivity and crosstalk

This paper develops the theoretical framework to understand the capability of the interferometric surface plasmon microscope to quantify sample properties in a confined region. We use rigorous diffraction theory to quantify the ability of the system to measure local properties and eliminate crosstalk from adjacent regions. We argue that the interferometric system in the defocused condition defines the measured point of excitation and reradiation of the surface plasmons; which greatly improves localisation. We also present results for the noninterferometric microscope, which confirm that the interferometric based system can perform quantitative measurements over smaller regions.

Confocal surface plasmon microscopy with pupil function engineering

Surface Plasmon microscopy can measure local changes of refractive index on the micron scale. Interferometric plasmon imaging delivers quantitative high spatial resolution sensitive to refractive index. In addition the so called V(z) method allows image contrast to be controlled by varying the sample defocus without substantially degrading spatial resolution. Here, we show how a confocal system provides a simpler and more stable alternative. This system, however, places greater demands on the dynamic range of the system. We therefore use a spatial light modulator to engineer the microscope pupil function to suppress light that does not contribute to the signal.

Scanning and non-scanning surface plasmon microscopy to observe cell adhesion sites

We observe adhesion sites of a cell on a substrate with high resolution. Since this observation requires interfacial measurements between the cell and the substrate, we employ scanning localized surface plasmon microscopy. We experimentally show that focal adhesion sites of a mouse muscle cell can be observed without fluorescent labeling. We also show that a non-scanning surface plasmon microscope combined with the scanning localized surface plasmon microscope contributes to observing an entire cell adhesion site and identify regions of interest.

Bragg-Scattered Surface Plasmon Microscopy: Theoretical Study

We present a new approach to surface plasmon microscopy with high refractive index sensitivity and spatial resolution that is not limited by the propagation length of surface plasmons. It is based on a nanostructured metallic sensor surface supporting Bragg-scattered surface plasmons. We show that these non-propagating surface plasmon modes are excellently suited for spatially resolved observations of refractive index variations on the sensor surface owing to their highly confined field profile perpendicular to as well as parallel to the metal interface. The presented theoretical study reveals that this approach enables reaching similar refractive index sensitivity as regular surface plasmon resonance (SPR) microscopy and offers the advantage of improved spatial resolution when observing dielectric features with lateral size <10 μm for the wavelength around 800 nm and gold as the SPR-active metal. This paper demonstrates the potential of Bragg-scattered surface plasmon microscopy for high-throughput SPR biosensing with high-density microarrays.

Amplitude and phase images of cellular structures with a scanning surface plasmon microscope

Imaging cellular internal structure at nanometer scale axial resolution with non invasive microscopy techniques has been a major technical challenge since the nineties. We propose here a complement to fluorescence based microscopies with no need of staining the biological samples, based on a Scanning Surface Plasmon Microscope (SSPM). We describe the advantages of this microscope, namely the possibility of both amplitude and phase imaging and, due to evanescent field enhancement by the surface plasmon resonance, a very high resolution in Z scanning (Z being the axis normal to the sample). We show for fibroblast cells (IMR90) that SSPM offers an enhanced detection of index gradient regions, and we conclude it is very well suited to discriminate regions of variable density in biological media such as cell compartments, nucleus, nucleoli and membranes.

Enhanced detection of fluorescent nanospheres using two-channel radially polarized surface plasmon microscopy

We detected single dye-stained latex nanospheres as small as 20 nm using a two-detection channel modified surface plasmon microscope. We found that a radially polarized incident beam leading to excitation of well-focused surface plasmons induces both fluorescence and elastic linear scattering from the spheres. The two complementary emitted signals were detected in parallel by the two separated channels, leading to well-colocalized images. We obtained high spatial resolutions for both channels down to 250 nm in the lateral direction and 300 nm along the longitudinal axis. We believe this multimodal microscope can be useful to track nano objects and to compensate for intermittent fluorescence, thanks to a permanently activated parallel scattering detection channel.

In situ imaging of micropatterned phospholipid membranes by surface plasmon fluorescence microscopy

A parallel microscopic imaging technique, surface plasmon microscopy (SPM)–surface plasmon fluorescence microscopy (SPFM), is introduced as a versatile analytical tool to monitor biochips. In spite of the fact that the fluorescence excited by surface plasmon is 1–2 order stronger compared with the total internal reflection fluorescence microscopy, SPFM has not fully utilized its advantages because fluorescence from fluorophores near the gold surface is almost entirely quenched due to the Förster energy transfer. In this study, SiO 2 layer sputtered on the gold substrate suppressed the quenching of fluorescence and enabled a parallel measurement of SPM and SPFM. As a model system, micropatterned membranes composed of polymeric and fluid phospholipid bilayers were employed. The difference of film thickness could be detected by SPM, and SPFM provided information on the composition and structure of membranes, enabling the distinction between polymeric and fluid phospholipid bilayers. These results suggest the general applicability of SPM–SPFM for various formats of biochips.

Modeling of the scanning surface plasmon microscope

We propose a family of exact solutions of Maxwell's equations to model some aspects of the imaging process involved in the scanning surface plasmon microscope (SSPM). More precisely, we compute the SSPM response of a spherical nanoparticle immobilized close to a thin gold layer and illuminated by a tightly focused spot. We discuss the influence of parameters such as the defocus and the width of the gold layer on the image contrast. We show that this microscopy combines a subwavelength spatial resolution together with high sensitivity to small changes in dielectric properties on the nanoparticle.

High resolution imaging of patterned model biological membranes by localized surface plasmon microscopy

We report on microscopic imaging of phospholipid membranes. To achieve nonlabel, noncontact, and high spatial resolution imaging of the membranes, we use optically excited localized surface plasmons as a virtual measurement probe to obtain the local refractive index. This enables significantly higher lateral resolution of approximately 170 nm. We reveal that the developed microscope has the capability of observing lipid bilayers with thickness of 3.0 nm deposited into the gaps in a patterned lipid bilayer with thickness of 4.6 nm. We find that the thickness resolution against the deposited lipid bilayer is approximately 0.33 nm.

Image Formation in Wide-Field Microscopes Based on Leakage of Surface Plasmon-Coupled Fluorescence

<para xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> A proof-of-concept imaging technique that combines the advantages of wide-field surface plasmon, leakage radiation, and total internal reflection fluorescence microscopy methods is presented. High-contrast non-scanning images with subwavelength resolution of patterned and homogeneous samples coated with a fluorescent material were demonstrated. We show that the image formed in the back focal plane of the objective lens can be reconstructed from the image of the sample's surface using an algorithm similar to that used in computerized tomographic imaging. Our experimental results suggest that there is a 1-D Fourier transform relationship between any line amplitude profile at a given angle passing through the center of the sample's surface image and a line amplitude profile passing through the center of the back-focal-plane image at the same angle. </para>

Polarization conversion in confocal microscopy with radially polarized illumination

The effects of using radially polarized illumination in a confocal microscope are discussed, and the introduction of a polarization mode converter into the detection optics of the microscope is proposed. We find that with such a configuration, bright-field imaging can be performed without losing the resolution advantage of radially polarized illumination. The detection efficiency can be increased by three times without having to increase the pinhole radius and sacrificing the confocality of the system. Furthermore, the merits of such a setup are also discussed in relation to surface plasmon microscopy and single-molecule orientation studies, where the doughnut point spread function can be engineered into a single-lobed point spread function.

Localized surface plasmon microscope with an illumination system employing a radially polarized zeroth-order Bessel beam

We propose an imaging principle that employs a radially polarized zeroth-order Bessel beam in the illlumination system of the localized surface plasmon microscope. The illumination system enables the microscope to visualize a refractive index distribution on a substrate fabricated in the Kretschmann configuration by the measurement of reflected intensity. The experimentally observed image of a particle reveals that the spatial resolution reaches the optical diffraction limit. The proposed principle can contribute to increase the imaging speed of localized surface plasmon microscopy by use of a beam scanning device.

Radial polarization induced surface plasmon virtual probe for two-photon fluorescence microscopy

Surface plasmons excited by a focused femtosecond radially polarized beam on a metal surface form a standing wave pattern with a sharp peak that can be used as a "virtual probe" for surface plasmon microscopy. The rotational symmetry of radially polarized light effectively provides the TM polarization required for coupling to the surface plasmons while the short pulse nature of the probe allows for nonlinear processes to be studied.

Towards A New Generation Of Single Molecule High Resolution Sensors

We present here the methodology that we have developed in the transdisciplinary Joliot Curie laboratory in partnership with the laboratory of physics of Ecole Normale Superieure Lyon (France) during the past four years for the preparation of sensor chips allowing the detection of single biomolecule interactions (DNA-DNA, DNA-proteins). Three confined measurement methods have been implemented, namely atomic force microscopy in liquid media which is used to capture the structural and mechanical properties of DNA and DNA-protein assemblies, interfacial impedance spectroscopy which probes the surface resistivity and capacitance of adsorbed DNA or lipidic layers, and high resolution surface plasmon microscopy which allows the detection of single biological molecules or assemblies of nanometer size such as DNA plasmids, DNA protein complexes (nucleosomes). Combining these three techniques we have afforded the possibility of direct in-situ synthesis and characterization of DNA-protein complexes such as nucleosomes, and the dynamical study of their stability in time, depending both on DNA sequence and its mechanical properties and on the ionic strength, buffer composition and pH of the liquid medium in contact with these complexes. The possibility of recording the dynamical response in liquid medium of a single molecular assembly under an external constraint modification, without need of a fluorescent marking is very challenging in the domain of biosensors since it should allow a definite improvement of the sensitivity and selectivity of DNA and protein sensor chips. We will discuss this assertion, based on experimental evidences and theoretical estimations of the sensitivity limits of the three experimental methods undertaken in this project.