Super-Resolution Dynamic Imaging of Dendritic Spines Using a Low-Affinity Photoconvertible Actin Probe

The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution

Caractérisation d'impulsions brèves. Mise en forme temporelle et Spectrale pour une application à l'endomicroscopie bi-photonique

This work first of all relates to two original techniuqes od single shot temporal characterization of short light pulses. A liquid core fibre autocorrelator based on two photon fluorescence is demonstrated. The second method, named SPIRIT is based on spectral interferometry resolved in time. It implements an interferometric stage followed by a non-linear stage of all optical time sampling. A two dimensional evolution of SPIRIT benefiting from spectral and temporal dimensions is also presented.The second part of this work relates to femtosecond pulse delivery with optical fibers for an application to non-linear endomicroscopy. TEmporal and spectral shaping allows the compensation for chromatic dispersion and self phase modulation occuring in the endoscopic fiber that is made of a bundle of thousands of optical fibers. The non-linear endomicrsocope allowed the recording of two photon images of human colon cells for a low average power incident on the biological tissues.Ce travail concerne tout d'abord deux techniques originales de caractérisation monocoup d'impulsions lumineuses brèves. un autocorrélateur à deux photons à fibre optique à coeur liquide fluorescent est tout d'abord présenté. La seconde méthode, nommée SPIRIT, s'appuie sur l'interférométrie spectrale à décalage résolue temporellement sans référence. Elle met en oeuvre une étape interférométrique suivie d'une étape non-linéaire d'échantillonnage temporel tout optique. Une évolution bi-dimensionnelle de SPIRIT profitant des dimensions spectrale et temporelle et également démontrée.La seconde partie de ce travail concerne une technique d'acheminement d'impulsions femtosecondes énergétiques par fibre optique en vue d'une application à l'endomicroscopie non-linéaire. La mise en forme temporelle et spectrale du signal permet de pré-compenser la dipersion chromatique et l'automodulation de phase se produisant dans la fibre endoscopique constituée d'un faisceau de milliers de fibres optiques. L'endomicroscope non-linéaire a permis l'enregistrement d'images bi-photoniques de cellules de colon humain pour une puissance moyenne faible

Beside FROG and SPIDER, a new single shot fully deterministic approach: SPIRIT

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Time resolved spectral interferometry for single shot femtosecond characterization

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Single shot Spectral shearing Interferometry for real time femtosecond pulse field reconstruction

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Fractional-order Fourier analysis for ultrashort pulse characterization

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