Pierre Jouchet

L’imagerie tridimensionnelle par localisation de molécules uniques (SMLM) permet d’obtenir des résolutions de quelques dizaines de nanomètres mais présentent encore certaines limitations liées notamment à une précision axiale non uniforme et une profondeur d’observation souvent limitée au premier micron de l’échantillon. Nous proposons ici une nouvelle approche de localisation de molécules uniques, appelée ModLoc, qui repose sur la modulation et la démodulation du signal de fluorescence des molécules uniques via l’utilisation d’une excitation structurée et modulée temporellement. Dans un premier temps, nous exposons les fondamentaux de l’imagerie SMLM et les limites actuelles de ce domaine. Les subtilités de ce nouveau principe de localisation sont ensuite détaillées et montrent un gain de précision théorique d’un facteur 3. Des études du caractère temporel aléatoire de l’émission des sondes fluorescentes en imagerie SMLM révèlent la nécessité d’intégrer des solutions optiques rapides (proche du kHz). La validation expérimentale du gain en précision accessible est démontrée grâce à la mise en place de deux dispositifs optiques. Nous choisissons d’appliquer ce principe afin d’améliorer la précision de localisation axiale des molécules fluorescentes. Les résultats obtenus mettent en évidence une précision de localisation uniforme de 7.5 nm et jusqu’à 7 microns en profondeur sur des échantillons de calibrations et des échantillons biologiques. La robustesse de la méthode pour l’imagerie SMLM en profondeur est également démontrée grâce notamment à des acquisitions effectuées à 30 µm de profondeur dans des milieux aberrants. Différentes pistes d’amélioration du dispositif actuel ainsi que l’extension de cette approche de localisation modulée à l’observation d’autres grandeurs telle que le temps de vie et l’orientation des molécules fluorescentes sont proposées.

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

Localisation microscopy of individual molecules allows one to bypass the diffraction limit, revealing cellular organisation on a nanometric scale. This method, which relies on spatial analysis of the signal emitted by molecules, is often limited to the observation of biological objects at shallow depths, or with very few aberrations. The introduction of a temporal parameter into the localisation process through a time‐modulated excitation was recently proposed to address these limitations. This method, called ModLoc, is demonstrated here with an alternative flexible strategy. In this implementation, to encode the time‐modulated excitation a digital micromirror device (DMD) is used in combination with a fast demodulation approach, and provides a twofold enhancement in localisation precision.Layout: Nowadays, we can use an optical microscope to observe how proteins are organised in 3D within a cell at the nanoscale. By carefully controlling the emission of molecules in both space and time, we can overcome the limitations set by the diffraction limit. This allows us to pinpoint the exact location of molecules more precisely. However, the usual spatial analysis method limits observations to shallow depths or causing low distortion of optical waves.To overcome these restrictions, a recent approach introduces a temporal element to the localisation process. This involves changing the illumination over time to enhance the precision of localisation. This method, known as ModLoc, is showcased here using a flexible and alternative strategy. In this setup, a matrix of micrometric mirrors, working together with a fast demodulation optical module, is used to encode and decode the time‐modulated information. This combination results in a twofold improvement in localisation precision.

Structured illumination in single-molecule localization microscopy provides new information on the position of molecules and thus improves the localization precision compared to standard localization methods. Here, we used a time-shifted sinusoidal excitation pattern to modulate the fluorescence signal of the molecules whose position information is carried by the phase and recovered by synchronous demodulation. We designed two flexible fast demodulation systems located upstream of the camera, allowing us to overcome the limiting camera acquisition frequency and thus to maximize the collection of photons in the demodulation process. The temporally modulated fluorescence signal was then sampled synchronously on the same image, repeatedly during acquisition. This microscopy, called ModLoc, allows us to experimentally improve the localization precision by a factor of 2.4 in one direction, compared to classical Gaussian fitting methods. A temporal study and an experimental demonstration both show that the short lifetimes of the molecules in blinking regimes impose a modulation frequency in the kilohertz range, which is beyond the reach of current cameras. A demodulation system operating at these frequencies would thus be necessary to take full advantage of this new localization approach. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.