Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK)

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors

Sonde opto-mécaniques pour la microscopie AFM rapide

National audienceIn the field of microscopy, the atomic force microscope (AFM) invented in 1986 was brought little, but nonetheless impressive, incremental developments since then. This instrument’s performances, and in particular imaging speed, are mainly limited by its cantilever-type force probe whose resonance frequency peaks at a few MHz. This thesis work presents a new concept of AFM probe, an optomechanical (OM) one, and custom instrument’s components to exploit its performances. Indeed, the 100+ MHz vibrating OM probes tested in this manuscript demonstrate an exquisite thermomechanical limit of detection of 4.5x1E-17 m/√Hz at room temperature, lower than any other AFM probe detection, and an instrument-limited 10 pm vibration amplitude. This OM probe consists of a suspended silicon ring with a 10 µm radius, acting as a mechanical resonator and a whispering-gallery-mode optical resonator. The two are intimately coupled by the ring shape: when the ring vibrates in a breathing mode, the optical cavity length varies and so does its resonance wavelength around its central value 1.55 µm. Characterization of numerous OM probes with different designs are investigated to find optimal designs, that is a 100 nm to 200 nm evanescent-coupling-gap and spokes width below 100 nm. Through deep characterization, acute phenomenon is also highlighted as the super-mode. Two alternatives to put the probe in vibration are compared: capacitive and optical. Stability and noise study of the probe help identify an additional noise source in optical actuation, that seem to be related to the optical background signal. Each developed component of the AFM instrument is detailed from piezoelectric scanner to data acquisition and processing. Because of a fabrication technological lock, the tip of the OM probe could not approach any surface as it did not protrude from the substrate on which the probe is made. A conventional AFM lever is therefore used to interact mechanically with the AFM probe. The instrument’s bandwidth is then characterized in operation, demonstrating a state-of-the-art 100 kHz feedback-loop bandwidth. Finally, a first pseudo-image is achieved with such probes, demonstrating the whole instrument operation.Dans le domaine de la microscopie, le microscope à force atomique (AFM), inventé en 1986, est aujourd’hui toujours basé sur le même concept de sonde de force : le levier. Les performances de l’AFM, et en particulier sa vitesse d'imagerie, sont principalement limitées par ce levier, dont la fréquence de résonance plafonne à quelques MHz. Ce travail de thèse présente un nouveau concept de sonde AFM, une sonde optomécanique (OM), ainsi que les développements sur l’instrument pour exploiter ses performances. En effet, des sondes OM vibrant à plus de 100 MHz sont développées et exploitées dans ce manuscrit. Elles démontrent une limite de détection thermomécanique remarquable de 4.5x1E-17 m/√Hz à température ambiante, inférieure à celle de toute autre sonde AFM, permettant un fonctionnement avec une amplitude de vibration de 10 pm. Cette sonde OM est constituée d'un anneau de silicium suspendu d'un diamètre de 20 µm, agissant à la fois comme un résonateur mécanique et un résonateur optique à mode de galerie. Les deux sont intimement couplés par la forme de l'anneau : lorsque l'anneau vibre dans un mode de respiration, la longueur de la cavité optique varie et sa longueur d'onde de résonance varie autour de la longueur d’onde centrale de 1,55 µm. De nombreuses variantes de sondes OM sont caractérisées pour trouver la conception optimale, conduisant à un gap de couplage évanescent de 100 nm à 200 nm et une largeur de rayons de suspension inférieure à 100 nm. Grâce à une caractérisation approfondie, un phénomène singulier est également mis en évidence : le super-mode. Deux alternatives pour mettre la sonde en vibration sont comparées : l’actionnement capacitif et optique. L'étude de la stabilité et du bruit de la sonde permet d'identifier une source de bruit supplémentaire en actionnement optique. Ensuite, les sondes OM sont intégrées dans un instrument AFM dont chaque composant est spécialement développé, du scanner piézoélectrique à l'acquisition et au traitement des données. À cause d’un verrou technologique de fabrication, la pointe de la sonde OM n’a pas pu être approchée d’une surface : elle ne dépasse pas du substrat sur lequel la sonde est fabriquée. Un levier AFM classique est donc utilisé pour interagir mécaniquement avec la sonde AFM. La bande passante de l'instrument est alors caractérisée en fonctionnement, démontrant une bande passante de boucle de rétroaction de 100 kHz, à l’état de l’art. Enfin, une première pseudo-image est réalisée avec ces sondes, démontrant le fonctionnement complet de l'instrument

Opto-mechanical probe for high speed AFM microscopy

Dans le domaine de la microscopie, le microscope à force atomique (AFM), inventé en 1986, est aujourd’hui toujours basé sur le même concept de sonde de force : le levier. Les performances de l’AFM, et en particulier sa vitesse d'imagerie, sont principalement limitées par ce levier, dont la fréquence de résonance plafonne à quelques MHz. Ce travail de thèse présente un nouveau concept de sonde AFM, une sonde optomécanique (OM), ainsi que les développements sur l’instrument pour exploiter ses performances. En effet, des sondes OM vibrant à plus de 100 MHz sont développées et exploitées dans ce manuscrit. Elles démontrent une limite de détection thermomécanique remarquable de 4.5x1E-17 m/√Hz à température ambiante, inférieure à celle de toute autre sonde AFM, permettant un fonctionnement avec une amplitude de vibration de 10 pm. Cette sonde OM est constituée d'un anneau de silicium suspendu d'un diamètre de 20 µm, agissant à la fois comme un résonateur mécanique et un résonateur optique à mode de galerie. Les deux sont intimement couplés par la forme de l'anneau : lorsque l'anneau vibre dans un mode de respiration, la longueur de la cavité optique varie et sa longueur d'onde de résonance varie autour de la longueur d’onde centrale de 1,55 µm. De nombreuses variantes de sondes OM sont caractérisées pour trouver la conception optimale, conduisant à un gap de couplage évanescent de 100 nm à 200 nm et une largeur de rayons de suspension inférieure à 100 nm. Grâce à une caractérisation approfondie, un phénomène singulier est également mis en évidence : le super-mode. Deux alternatives pour mettre la sonde en vibration sont comparées : l’actionnement capacitif et optique. L'étude de la stabilité et du bruit de la sonde permet d'identifier une source de bruit supplémentaire en actionnement optique. Ensuite, les sondes OM sont intégrées dans un instrument AFM dont chaque composant est spécialement développé, du scanner piézoélectrique à l'acquisition et au traitement des données. À cause d’un verrou technologique de fabrication, la pointe de la sonde OM n’a pas pu être approchée d’une surface : elle ne dépasse pas du substrat sur lequel la sonde est fabriquée. Un levier AFM classique est donc utilisé pour interagir mécaniquement avec la sonde AFM. La bande passante de l'instrument est alors caractérisée en fonctionnement, démontrant une bande passante de boucle de rétroaction de 100 kHz, à l’état de l’art. Enfin, une première pseudo-image est réalisée avec ces sondes, démontrant le fonctionnement complet de l'instrument.In the field of microscopy, the atomic force microscope (AFM) invented in 1986 was brought little, but nonetheless impressive, incremental developments since then. This instrument’s performances, and in particular imaging speed, are mainly limited by its cantilever-type force probe whose resonance frequency peaks at a few MHz. This thesis work presents a new concept of AFM probe, an optomechanical (OM) one, and custom instrument’s components to exploit its performances. Indeed, the 100+ MHz vibrating OM probes tested in this manuscript demonstrate an exquisite thermomechanical limit of detection of 4.5x1E-17 m/√Hz at room temperature, lower than any other AFM probe detection, and an instrument-limited 10 pm vibration amplitude. This OM probe consists of a suspended silicon ring with a 10 µm radius, acting as a mechanical resonator and a whispering-gallery-mode optical resonator. The two are intimately coupled by the ring shape: when the ring vibrates in a breathing mode, the optical cavity length varies and so does its resonance wavelength around its central value 1.55 µm. Characterization of numerous OM probes with different designs are investigated to find optimal designs, that is a 100 nm to 200 nm evanescent-coupling-gap and spokes width below 100 nm. Through deep characterization, acute phenomenon is also highlighted as the super-mode. Two alternatives to put the probe in vibration are compared: capacitive and optical. Stability and noise study of the probe help identify an additional noise source in optical actuation, that seem to be related to the optical background signal. Each developed component of the AFM instrument is detailed from piezoelectric scanner to data acquisition and processing. Because of a fabrication technological lock, the tip of the OM probe could not approach any surface as it did not protrude from the substrate on which the probe is made. A conventional AFM lever is therefore used to interact mechanically with the AFM probe. The instrument’s bandwidth is then characterized in operation, demonstrating a state-of-the-art 100 kHz feedback-loop bandwidth. Finally, a first pseudo-image is achieved with such probes, demonstrating the whole instrument operation

Low Latency Demodulation for High-Frequency Atomic Force Microscopy Probes

International audienceOne prerequisite for high-speed imaging in dynamic-mode atomic force microscopy (AFM) is the fast demod-ulation of the probe signal. In this contribution, we present the amplitude and phase estimation method based on the acquisition of four points per oscillation, with the sampling frequency being phase-locked on the probe actuation. The method is implemented on a RedPitaya platform, with its clock being generated from the actuation signal of the probe. Experimental characterizations using square-modulated sine waves show that latency of 500 ns is achieved with a carrier frequency of 10 MHz, which is ten times faster compared with a state-of-the-art lock-in amplifier. A tracking bandwidth greater than 200 kHz is obtained experimentally. The method is eventually applied to a close-loop AFM scan realized using a 15-MHz AFM probe, showing its suitability for high-frequency oscillating probes. Index Terms-Amplitude and phase demodulation, atomic force microscopy (AFM), field-programmable gate array (FPGA) implementation

High Speed Atomic Force Microscope

International audienceAtomic Force Microscope (AFM) is now a common tool for material analysis in the academic and industrial areas because it enables non-destructive high-resolution images of nanometric objects. However, a main drawback is the slow scan rate that hinders many potential applications. Recently, breakthroughs have been achieved in AFM sensors based on MEMS technology, allowing to extend AFM operation in terms of measurement bandwidth and data acquisition. The present work focusses on developing an electronic controller for AFM featuring the wide bandwidth and the fast data processing rate required to enable the exploitation of the full potential of MEMS AFM sensors

Optomechanics: a key towards next-generation experiments in atomic force microscopy?

International audienceAtomic Force Microscopy (AFM) is a versatile and ubiquitous technique based on a resonating tip probe that interacts with the sample to be analysed in terms of topography and mechanical properties. Next-generation investigations in molecular biophysics like protein folding/unfolding, receptor-ligand interaction and molecular diffusion on cell membrane, require tracking molecular forces at the nanosecond timescale in a non-perturbative manner [1]. Conventional AFM probes, made of micro-cantilevers vibrating in the MHz range with nanometer amplitudes and combined with an optical deflection detection system, are the current bottleneck to reach the required performances. Both aspects, a very low and non-perturbative vibration amplitude, time resolution, and measurement bandwidth, are impacted by the same chief parameter: the frequency f of the probe mechanical resonator. While a higher frequency unlocks the bandwidth and the time resolution, it also sets the Brownian motion low enough to provide exquisite signal-to-noise ratio and force resolution even for vibration amplitude in the picometer range, i.e. much lower than the molecular dimensions. Recent advances in optomechanical devices technology allow tackling the challenge by offering resonators at very high frequencies greater than the GHz and unprecedented motion sensitivity below 10-17 m.Hz-0.5. The talk will present our recent developments introducing a fully-optically operated resonating optomechanical AFM probe above 100 MHz of frequency, 2 decades above the fastest commercial cantilever probes, while Brownian motion 4 orders below [2]. Based on a silicon technology and operated at 1.55 µm wavelength, the probe shown in Fig. 1 demonstrates high-speed sensing of mechanical interactions with a sub-picometer resonantly driven motion, breaking open current locks for faster and finer force spectroscopy at the molecular level. Figure 1. Scanning electron microscopy image of the optomechanical atomic force microscopy probe. The ring-shaped whispering gallery mode resonator is 20 µm in diameter. A 4 µm-long tip apex protrudes from the ring, aiming at sensing near-field forces when interacting with a surface.[1] H. Yu et al., Science, 355, 945-950, (2017)[2] P.E. Allain et al., arXiv:1810.06209, (2018

MEMS-based atomic force microscopy probes: from electromechanical to optomechanical vibrating sensors

International audienceScanning probe microscopy has been one of the most important instrumental discoveries during the last quarter of the last century. In particular, atomic force microscopy (AFM) is a cross-disciplinary technique able to provide sample morphology down to the atomic scale. It offers invaluable tools to support the development of nano-sciences, information technologies, micro-nanotechnologies and nano-biology. For more than 20 years, boosting the scan rate of AFM has been an increasingly important challenge of the community. However still today, performing routine and user-friendly AFM experiments at video rate remains unreachable in most cases. The conventional AFM probe based on a micro-sized vibrating cantilever is the major obstacle in terms of bandwidth and resonance frequency.Following a brief description of the context of the work, the talk will first describe the development of AFM probes based on MEMS devices that make use of ring-shaped microresonators vibrating above 10 MHz. A focus will be dedicated to the electrical detection scheme. Based on capacitive transduction and microwave reflectometry, it achieves a displacement resolution of 1E-15 m/√Hz, allowing the measurement of the thermomechanical vibration of the MEMS AFM probes in air. Imaging capability obtained on DNA origamis samples at a frame rate greater than 1 image/s will be shown as well as investigation of block copolymer surfaces to elucidate the tip-surface interaction when vibration amplitudes are lower than 100 pm.In the following, our recent research direction at the convergence of the fields of micro/nanosystems and VLSI optomechanics on silicon chips will be presented. Optomechanical resonators allow indeed overcoming the resolution limitation imposed by usual electromechanical transduction schemes. Here, we will introduce fully optically driven and sensed optomechanical AFM probes which resonance frequency is above 100 MHz and Brownian motion below 1E-16 m/√Hz, paving the way for high-Speed AFM operation with exquisite resolutions at sub-angstrom vibration amplitudes