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Recherche

Recherche

1) Photothermal Interference Contrast (PIC) Method

Ambient optical detection of labeled molecules is limited for fluorescent dyes by photobleaching and for semiconducting nanoparticles by blinking effects. Because nanometer-sized metal particles do not optically bleach, they may be useful optical labels if suitable detection signals can be found. We are now able to perform far-field optical detection of gold colloids down to diameters of 2.5 nanometers with a photothermal method (Photothermal Interference Contrast, PIC) that combines high-frequency modulation and polarization interference contrast. The photothermal image is immune to the effects of scattering background, which limits particle imaging through Rayleigh scattering to diameters larger than 40 nanometers.

2) Photothermal Heterodyne Imaging (PHI)

The sensitivity of the PIC technique, though high, is limited. In particular, when high NA objectives are used, depolarization effects degrade the quality of the overlap between the two arms of the interferometer. This effect is even more serious when NPs are imaged in cellular environments. As a consequence, quite high laser beam intensities were used for the detection of individual 10 nm gold NP in cells, restricting the use of the PIC method to fixed samples.

We have recently developed another photothermal based method, Photothermal Heterodyne Imaging (PHI)Berciaud. Its sensitivity lies two orders of magnitude above earlier methods and it allowed for the unprecedented detection of individual 1.4 nm (67 atoms) gold NPs. For a given NP size, it also allows the use of much lower heating beam intensities than PIC. PHI uses a combination of a time-modulated heating beam and a non-resonant probe beam. The probe beam produces a frequency shifted scattered field as it interacts with the time-modulated variations of the refraction index around the absorbing NP. The scattered field is then detected through its beatnote with the probe field which plays the role of a local oscillator as in any heterodyne technique and can be extracted by lock-in detection.

The absorption spectroscopy of individual gold nanoparticles as small as 5 nm as recently been performed owing to PHI revealing intrinsic size effect in their optical response.

PHI was also used to record the first room temperature absorption spectra of single CdSe/ZnS semiconductor nanocrystals. Comparison with the photoluminescence spectra of each individual nanocrystal leads to the measurement of spectral Stokes shifts free from ensemble averaging. Since the photothermal absorption spectra are recorded in a high intensity cw excitation regime, the observed bands are assigned to transitions involving biexciton and trion states.

3) applications to biology

Single metallic nanoparticles imaging in cells by PIC.

In addition to its intrinsic sensitivity, the PIC images are remarkably insensitive to scattering background, even when arising from such strong scatterers as 300 nm latex beads (Boyer et al 2002). Furthermore, it is possible to image gold colloids in thick samples (e.g. cells) with a modified PIC setup. We performed the imaging of receptor proteins stained with individual 10 nanometer size gold particles in the plasma membrane of COS7 cells. The resolution in 3D of the PIC method is comparable to confocal setups and was also derived and confronted to measurements.

Single Nanoparticle Photothermal Tracking (SNaPT) of 5 nm gold beads in live cells

Tracking individual nano-objets in live cells during arbitrary long times is an ubiquitous need in modern biology. We applied PHI that we alternatively call Laser Induced Scattering around a NanoAbsorber (LISNA) as mean to track individual 5 nm gold nanoparticles on live cells. The key point for recording trajectories at video rate was the use of a triangulation procedure. The effectiveness of the method was tested against Single fluorescent Molecule Tracking in live COS7 cells on subsecond time scales. We further demonstrated recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. SNaPT has the unique potential to record arbitrary long trajectory of membrane proteins using non-fluorescent nanometer sized labels.

Optical readout of gold nanoparticles-based DNA microarrays without silver enhancement

LISNA can be used as novel readout scheme for gold nanoparticle-based DNA microarrays. It provides direct counting of individual nanoparticles present on each array spot and stable signals, without any silver enhancement. Given the detection of nanometer-sized particles, which minimize the steric hindrance, the linear dynamic range of the method is particularly large and well suited for microarray detection.

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