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.
We present the implementation of a fast wide-field optical sectioning technique called HiLo microscopy on a
fluorescence lifetime imaging microscope. HiLo microscopy is based on the fusion of two images, one with
structured illumination and another with uniform illumination. Optically sectioned images are then digitally
generated thanks to a fusion algorithm. HiLo images are comparable in quality with confocal images but they
can be acquired faster over larger fields of view. We obtain 4D imaging by combining HiLo optical sectioning,
time-gated detection, and z-displacement. We characterize the performances of this set-up in terms of 3D spatial
resolution and time-resolved capabilities in both fixed- and live-cell imaging modes.
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 NA<sub>obj </sub><
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.
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.
We present the development of a time resolved TIRF microscope illuminated by a
supercontinuum laser source. It permits to perform wide-field fluorescence lifetime imaging of
neurobiological processes at the plasma membrane with subwavelength axial resolution.
Total internal reflection fluorescence microscopy (TIRFM) is a powerful optical technique to observe
fluorescence close to surfaces. Associated with fluorescence lifetime imaging, TIRFM enables to measure
contrasts independent of fluorophores concentration and reveal intracellular activity with subwavelength axial
resolution. We developed an original setup which allows, thanks to a wide-field time resolved detection, to
measure nanoseconds fluorescence lifetimes of membrane receptors in order to apprehend their signalisation
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.
Two-photon microscopy is a key method for biological and medical research on cells and tissues mainly due to the submicronic spatial resolution. Unfortunately in its conventional form, this technique leads to long time recording for three-dimensional and fluorescence lifetime imaging because it requires a single point laser scanning. The most suitable way to improve acquisition time is to illuminate the biological sample with several excitation points simultaneously. We thus present a time-resolved multifocal multiphoton microscope. Besides the advantage of preserving biological samples by reducing by a factor 64 the exposition time, this method keeps also the possibility of measuring both intensity and lifetime images of the samples.
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.
Urinary cytology is employed in diagnostic guidelines of bladder cancer in anatomo-pathological laboratories mostly for its ability to diagnose non detectable cancers using cystoscopy, but also because it is a non-invasive and non-constraining technique for a regular follow-up of the more exposed populations. The impossibility to detect such cancers is mainly due to their localization either in the bladder or in the upper urinary tract and the prostate. However, urinary cytology lacks sensitivity, especially for the detection of low grade low stage tumors due to inherent limitation of morphological criteria to distinguish low grade tumor cells from normal urothelial cells. For this purpose, we developed, in addition to urinary cytology, an original screening of these cytological slides by using spectrally-resolved and time-resolved fluorescence as a contrast factor, without changing any parameters in the cytological slide preparation. This method takes advantage of a femtosecond Ti:sapphire laser, continuously tunable in the spectral range 700-950 nm allowing the observation of most endogenous cellular chromophores by biphotonic excitation. A commercial confocal microscope was also used in the measurements allowing an excitation of the samples between 458 nm and 633 nm. We observed that the fluorescence emission is differentially distributed in normal and pathological urothelial cells. Spectral- and time-resolved measurements attested this difference over about one hundred cases which have been tested to confirm the high accuracy of this non-invasive technique.
Two photon microscopy is a powerful tool for cells or tissues imaging. However it
presents the drawback of being a laser-scanning technique leading to long acquisition time for fluorescence lifetime imaging. Thus it is commonly limited to intensity images that only give indications on location of fluorophores but hardly reports physico-chemical properties and interactions
between cells components.
To preserve biological samples from too long experiments and provide a more complete
spectroscopic tool we developed a time-resolved multifocal multiphoton microscope. This
setup allows us to speed up the acquisition while keeping the possibility to measure both
intensity and lifetime images for all multifocal points.
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.
Two photon microscopy is a powerful tool for cells or tissues imaging. However it presents the drawback of being a laser-scanning technique leading to long time acquisition for 3D images. To preserve biological samples from too long experiments and provide a more complete spectroscopic tool we developed a time-resolved multifocal multiphoton microscope. This setup allows us to speed up the acquisition and gives both intensity and lifetime images for all multifocal points.
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.
The fluorescence decay in fluorescence lifetime imaging (FLIM) is typically fitted to a multi-exponential model with discrete lifetimes. The interaction between fluorophores in heterogeneous samples (e.g. biological tissue) can, however, produce complex decay characteristics that do not correspond to such models. Although they appear to provide a better fit to fluorescence decay data than the assumption of a mono-exponential decay, the assumption of multiple discrete components is essentially arbitrary and often erroneous. The stretched exponential function (StrEF) describes fluorescence decay profiles using a continuous lifetime distribution as has been reported for tryptophan, being one of the main fluorophores in tissue. We have demonstrated that this model represents our time-domain FLIM data better than multi-exponential discrete decay components, yielding excellent contrast in tissue discrimination without compromising the goodness of fit, and it significantly decreases the required processing time. In addition, the stretched exponential decay model can provide a direct measure of the sample heterogeneity and the resulting heterogeneity map can reveal subtle tissue differences that other models fail to show.
We report a whole-field fluorescence imaging microscope that combines 3-D spatial resolution by optical sectioning, using structured illumination, with fluorescence lifetime imaging and spectrally-resolved imaging. We show the potential of this technique in the elimination of common artefacts in fluorescence lifetime imaging and apply it to study the dependence of the lifetime on the emission wavelength in biological tissue.
Acousto-optic imaging in strongly light-scattering tissues seeks to reveal optical contrasts in these turbid media. Nevertheless, this technique happens to be also sensitive to their acoustic contrasts. We have built a new setup combining a dedicated echograph and an acousto-optic imager in a single apparatus. Thanks to this setup, we have studied ultrasound absorbent and light absorbent features embedded in several centimeter thick biological tissues, and we have compared for the first time the acoustic and acousto-optic signals recorded in the same configuration. We show that even though optical contrast is the ultimate goal of this technique, preliminary acoustic investigation of the tissue is necessary to interpret correctly acousto-optical signals.
Acousto-optic imaging consists in tagging multi-scattered photon paths with a focused ultrasonic beam. This technique should give optical information on hidden structures in several centimeter thick scattering media, with a millimetric resolution. We have coupled our previous acousto-optic imaging setup with a suitably designed echograph. Thanks to a single 3 MHz multi-ring emitter, working either in pulsed or c.w. mode, we can get acoustic as well as acousto-optic responses of structures in biological tissues.
We have demonstrated the feasibility of tagging the photons trajectories with a focused ultrasonic field, to reveal optical contrast in biological tissues. 3D images have been obtained on real ex-vivo structures of animal as well as human tissues, through a thickness ranging from 2 cm to 4.5 cm. We are developing the coupling of this acousto-optical imaging with a traditional echograph.
Matrices of detectors carrying up to a few millions of pixels have changed in many aspects the science of imaging from astronomy to popular photography as well as from X rays to infrared. Most of the time they have been used for signal acquisition but rarely as an active part of the signal processing. A few years ago we have proposed ' touse such sensors, which convert radiations into charges and store them before reading, in order to achieve parallel lock-in or heterodyne detection at frequencies much higher than the speed of image acquisition. In the usual single channel lock-in detection scheme the (amplified) periodical noisy signal as multiplied by a noiseless synchronous reference signal and then filtered by a low-pass filter. In our approche 2 roughly speaking, the low-pass filter is replaced by the charge integration during one image acquisition whereas the multiplication is replaced by a synchronous excitation of the source (typically each half or quarter of period). Let us point out that the gain in term of signal-to-noise ratio is about the same in the two approaches (as long as we have to face a white noise such as the shot noise) but in the last one acquisition time can be i04 to iO times faster. In the two examples that we will describes below we will take advantage of the new promising field of biomedical imaging to demonstrate how helpful is our approach for imaging inside biological tissues which are strong light scatterers.
We describe here the detailed signal processing technique that we have introduced to detect ultrasonic speckle modulation using a CCD array. We show that this new approach leads to a better signal to noise ratio than the single detector one not only because all the speckle grains signals are used but also because averaging over a few 104 grains smoothes this random signal. Finally a few examples of signal generated by locally absorbing volume immersed in real biological tissues are given.
We have built an interference microscope that produces in real-time images of cross-section slices located at adjustable depths inside 3-D objects. The microscope is based on a Michelson-type polarization interferometer. A light emitting diode (LED), used as an optical source at (lambda) equals 840 nm with short coherence length, provides optical sectioning ability with better than 10 micrometer resolution in the depth dimension. By using high numerical aperture objective lenses (NA equals 0.95), the depth resolution can be improved to better than 1 micrometer, in good agreement with theory. Images can be produced at the rate of 50 per second using a multiplexed lock-in detection and MMX assembler-optimized calculation routines. Cross-section images inside an onion and at different depths in a multilayer silicon integrated circuit are presented.
We investigate a novel approach of the ultrasonic tagging of photons path in scattering media: instead of using a single detector associated with a conventional detection scheme to image biological phantoms, we take advantage of a more efficient scheme using a CCD camera and parallel processing lock-in detection to reveal optical contrasts in real biological tissues.