Microscopie tridimensionnelle à très haute résolution par tomographie par cohérence optique

Dans ce manuscrit est présentée l étude de nouvelles techniques de microscopie tridimensionnelle destinées à l imagerie à haute résolution (~ 1 m) de milieux biologiques. Ces techniques sont basées sur la microscopie par cohérence optique, dont le principe est d exploiter la faible longueur de cohérence d une source de lumière blanche dans un microscope interférométrique, pour réaliser des coupes tomographiques sans balayage et de manière non destructive d échantillons diffusants. Une autre méthode, ayant pour ambition de produire des images tomographiques de la présence de structures, avec une résolution spatiale dépassant la limite de diffraction (10-100 nm), a également été étudiée. Elle utilise des nano-particules d or comme sondes locales multiples pour explorer les contours des structures creuses internes à l échantillon.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Distinct Nanoscale Calcium Channel and Synaptic Vesicle Topographies Contribute to the Diversity of Synaptic Function

The nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (similar to 10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (similar to 50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses

Initiation of gastrulation in the mouse embryo is preceded by an apparent shift in the orientation of the anterior-posterior axis

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Calculated time evolution of the observation volume of STED nanoscopy (tE-PSF).

<p>Lateral (<i>r</i>) intensity distribution <i>h</i>(<i>t</i>,<i>r</i>) (A,B), the full-width-at-half-maximum (FWHM(<i>t</i>)) (C,D; black, left axis) and the peak signal <i>h</i>(<i>t</i>,0) (C,D; red, right axis) of the tE-PSF as a function of the time of the STED beam action (excitation at time 0) for the CW-STED (A,C) and P-STED (B,D) modality (A,B; left panel: original, un-normalized data; right panel: normalization to 1 for each time). The excitation intensity profile <i>h</i>_exc(<i>r</i>), the detection efficiency profile <i>h</i>_em(<i>r</i>) and the STED intensity profile <i>I</i>_sted(<i>r</i>) are computed using Fourier theory <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421-Leutenegger2" target="_blank">[39]</a>. Given <i>I</i>_sted(<i>r</i>), <i>h</i>_exc(<i>r</i>) and <i>h</i>_em(<i>r</i>) the time evolution of the observation volume <i>h</i>(<i>t</i>,<i>r</i>) is calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone.0054421.e004" target="_blank">Equation (4</a>). We assumed an oil immersion objective of 1.4 numerical aperture, <i>τ</i>₌3.4 ns, <i>T</i> = 1/80 MHz, <i>T</i>_STED = 300 ps, <i>λ</i>(excitation)₌635 nm, <i>λ</i>(STED)₌760 nm and <i>λ</i>(emission)₌670 nm, the same average powers for both modalities, i.e. ς^* = 4.8 and 200 for CW and pulsed mode, respectively, and a detection pinhole with a projected diameter of 500 nm in the sample space (0.9 × the Airy disc diameter).</p

Scanning gP-STED nanoscopy images.

<p>Scanning images of microtubule of fixed PtK2 cells immunolabeled with the dye KK114 (A) and ATTO647N (B): confocal (left), time-gated gP-STED (middle left) and non-gated P-STED (middle right) images, and normalized intensity profiles along the arrows marked in the P-STED images. Excitation: 635 nm, <i>f</i> = 76 Mhz, <i>P_exc</i>₌5 µW; STED: 760 nm, <i>f</i> = 76 Mhz, <i>P_STED</i>₌45 mW (a), 70 mW (b); gated detection: <i>T_g</i> = 500 ps. Pixel size: 20 nm. Scale bars: 1 µm.</p

Dependence of the confinement of the E-PSF of CW-STED and gCW-STED recordings on the fluorescence lifetime.

<p>Correlative plot of values pairs of intensity-weighted average lifetime <<i>τ</i>> (<i>i</i> = 3) and FWHM of the intensity profiles through images of individual FNDs. The solid lines show linear regression fits to the experimental data with slopes of −1.99, −0.70 and −0.41 for <i>T_g</i> = 0 ns, 5 ns and 10 ns, respectively. Imaging conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054421#pone-0054421-g003" target="_blank">Figure 3B</a>.</p