DAISY combined Supercritical Angle Fluorescence (SAF) detection in order to provide an absolute axial reference to a point spread function engineering approach . The dual view optical setup permits to introduce a strong astigmatism and decouple lateral and axial information, thus extending the axial performances without degrading the lateral precision. This technique provides 3D absolute information over a 1-µm capture range above the glass coverslip and an axial localization precision down to 15 nm with minimal loss of lateral resolution and little sensitivity to field aberrations. We will present sequential and simultaneous up to 3 color 3D imaging in cells.
We propose Modloc, a new single molecule localization strategy based on a time signature. By shifting a structured excitation pattern, it induces a time modulated emission of the fluorophores where the phase holds its position. An efficient demodulation optical assembly compatible with short emitters On-time has been designed. Performances of ModLoc, will be discussed, in particular its 2 fold lateral precision improvement and its robustness to optical aberrations. By applying the modulation in a tilted direction, we will show that an almost isotropic 3D localization (~11 nm) is obtained over the whole capture range up to 30 µm in depth
We present a new technique based on the self-interference of Supercritical Angle Fluorescence (SAF) emission in
order to perform full-field cell membrane imaging. We show that our Point Spread Function (PSF) engineering
technique allows us to obtain a 100 nm axial sectioning while conserving the original lateral resolution of the
microscope. The images are acquired using an optical module that can be connected to any fluorescent microscope
to simultaneously monitor in real time both the cell membrane and in-depth phenomena.
Circumventing the limit imposed by diffraction is a major issue in the instrumental development to realize finer
resolutions in biological samples. With STED microscopy, we exploit the molecular transitions of the fluorescent
marker to image well below the Rayleigh criterion. Also in combination with STED, we propose to use an
alternative technique for optically sectioning fluorescent emitters close to the water-glass interface by selectively
filtering the supercritical emission at the pupil plane. We discuss the instrumental development of such a system
and its combination with other imaging techniques.
In this paper, we discuss the possibility of making a super-axially-resolved image of a biological sample using
supercritical angle diffusion. This labeling-free approach is suitable to any microscope equipped with a NAobj <
1.33 microscope objective and can be used either for conventional intensity imaging or for quantitative phase
imaging. We expose some results on beads an cells showing the potential of this method.
Single biomolecule behaviour can reveal crucial information about processes not accessible by ensemble
measurements. It thus represents a real biotechnological challenge. Common optical microscopy approaches
require pico- to nano-molar concentrations in order to isolate an individual molecule in the observation
volume. However, biologically relevant conditions often involve micromolar concentrations, which impose a
drastic reduction of the conventional observation volume by at least three orders of magnitude. This
confinement is also crucial for mapping sub-wavelength heterogeneities in cells, which play an important role
in many biological processes. We propose an original approach, which couples Fluorescence Correlation
Spectroscopy (FCS), a powerful tool to retrieve essential information on single molecular behaviour, and
nano-fakir substrates with strong field enhancements and confinements at their surface. These
electromagnetic singularities at nanometer scale, called "hotspots", are the result of the unique optical
properties of surface plasmons. They provide an elegant means for studying single-molecule dynamics at
high concentrations by reducing dramatically the excitation volume and enhancing the fluorophore signal by
several orders of magnitude. The nano-fakir substrates used are obtained from etching optical fiber bundles
followed by sputtering of a gold thin-film. It allows one to design reproducible arrays of nanotips.
Active plasmonic devices are much promising for optical devices and circuits at the nanoscale. We show that
single nanoparticles coupled to metallic surfaces are good candidates for integrated components with
nanometric dimensions. The localized plasmon of the nanoparticle launches propagating surface plasmons in
the metallic thin film. Direct particle observation using leaky wave microscope geometry permits easy
detection through the interference of the direct transmitted excitation light and the surface plasmon leaky
mode. Investigations of the optical response of a nanoparticle deposited on metallic thin metal films reveals
unexpectedly high transmission of light associated to contrast inversion in the images.
We present a new imaging technique using surface-plasmon mediated fluorescence microscopy. It uses a similar configuration as standard prismless Total Internal Reflection Fluorescence Microscopy with an additional metallic thin film. In the case of a silver thin film we show that this technique offers many advantages: distance dependence emission filter for improved signal to noise ratio and enhanced molecular detection efficiency. This technique is of particular interest in membrane and adhesion imaging. We present real time images on live cells.
The last decade has witnessed momentous advances in fluorescence microscopy. The introduction of novel fluorescent markers, together with the development of original microscopy techniques, made it possible to study biomolecular interactions in living cells and to examine the structure and function of living tissues. The emergence of these innovative techniques had a remarkable impact on all the life sciences. However, many biological and medical applications involve the detection of minute quantities of biomolecules, and are limited by the signal weakness in common observation conditions. Here, we show that silver and gold-coated microscope
slides can be used as mirror substrates to efficiently improve detection sensitivity when fluorescence microscopy
is applied to micrometer-thick biological samples. We report a fourfold enhancement of the fluorescence signal
and a noticeable strengthening of the image contrast, when mirror substrates are used with standard air microscope
objectives. We demonstrate that metal-coated substrates provide the means to get sensitivity-enhanced fluorescence detection with dry optics, while keeping a wide field observation and a large depth of field. This is a crucial advantage for automated and high-throughput applications to cell and tissue diagnostic analysis.
Fluorescence microscopy has become the method of choice in the majority of life-science applications. We describe development and use of mirror slides to significantly enhance the fluorescence signal using standard air microscope objectives. This technique offers sufficient gain to achieve high-sensitivity imaging, together with wide field of observation and large depth of focus, two major breakthroughs for routine analysis and high-throughput screening applications on cells and tissue samples.
In our attempt to reveal highly localized field enhancements on random metallic films using near-field
scattering probe microscopy we experimentally demonstrated the existence of narrow peaks when using a
monochromatic illumination. In order to get a better understanding of the second harmonic generation taking
place on such films we have undertaken the same kind of near-field experiments using femtosecond lasers
sources with high peak power able to induce the non linear response. These lasers have a spectral bandwidth
associated with the pulse duration, which is in the femtosecond range. With such spectral broadening we have
observed, as expected, a spatial broadening of the peaks at ω, which spread over distances in the 100-500 nm
range. The behavior of the peaks is quite different at 2 ω: they are found to be always very well localized (~10
nm) despite of the polychromatic nature of the light; moreover there is no clear correlation between the peaks
position at ω and those at 2 ω. This observation indicates, as often underlined in non linear processes, that
coherent interactions involving a distribution of available frequencies in the lasers spectra take place. These
frequencies ωn, coherently induce second harmonic generation as long as ωn + ωm = 2 ω.
Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for DNA chips. In some cases, detection of very low molecular concentrations and precise localization of biomarkers are limited by the weakness of the fluorescence signal. We present a new method based on sample substrates that improve fluorescence detection sensitivity. These active substrates consist in glass slides covered with metal (gold or silver) and dielectric (alumina) films and can directly be used with common microscope set-up. Fluorescence enhancement affects both excitation and decay rates and is strongly dependant on the distance to the metal surface. Furthermore, fluorescence collection is improved since fluorophore emission lobes are advantageously modified close to a reflective surface. Finally, additional improvements are achieved by structuring the metallic layer. Substrates morphology was mapped by Atomic Force Microscopy (AFM). Substrates optical properties were studied using mono- and bi-photonic fluorescence microscopy with time resolution. An original set-up was implemented for spatial radiation pattern's measurement. Detection improvement was then tested on commercial devices. Several biomedical applications are presented. Enhancement by two orders of magnitude are achieved for DNA chips and signal-to-noise ratio is greatly increased for cells imaging.
We have developed a new detection scheme based on a scanning near-field optical microscope to image both the linear and the non-linear (e.g. second harmonic generation (SHG)) on surfaces with sub-wavelength resolution. The microscope we used here scatters the evanescent waves that contain sub-wavelength information with the apex of a metallic tip. The resolution of this microscope is directly given by the size of the radius of curvature of the metal tip end
(about 5 nm). Our set-up is applied to the optical study of crystalline films and random metal surfaces. Using thin SBN films
(Strontium Barium Niobate) we demonstrate that near-field optics is a surface sensitive measurement. The ability to perform high quality and highly resolved images is transposed to increase the resolution of imaging in the THz domain. It is also used in the visible domain on random metal films. Several studies have demonstrated that random metal surfaces show localization of the electric field on small area called "hot spots" where the electric field can exceed the applied field by several orders of magnitude. Position of the hot spots depends on film structure, on the polarization and wavelength of the illuminating laser beam. In addition, these random metal films are known to be the source of nonlinear optical effects. We are currently working to precisely locate the respective position of the linear and non-linear hot spots on silver.
Fluorescence is widely used as a spectroscopic tool or for biomedical imaging, in particular for
DNA chips. Nanostructured metallic substrates permit to locally enhance the fluorescence signal
which offer the possibility both to detect very small fluorophore concentrations and to trace
precisely the bio-markers. We have developed substrates made of silver or gold nanoparticles
covered with a spacer layer of alumina. Double metallic and dielectric gradients permit to directly
map the fluorescence enhancement factor and to determine the best condition for maximum
enhancement. One and two photons excitations are studied. Fluorescence enhancement reaches two
orders of magnitudes. Lifetime measurements reveal additional information on the decay channels
induced by the nanoparticle presence.
Fluorescence is widely used as a spectroscopic tool or for biomedical imaging. To extend these measurements to small concentrations or to fluorophores with very low quantum yield we have developed nanostructured substrates made of silver nanoparticles covered with a spacerlayer of alumina. Factors of about 200 are obtained for fluorescence enhancement with two photon excitation. Lifetime measurements reveal additional information on the decay channels induced by the nanoparticle presence.