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Automatic segmentation of nuclei in low-light microscopy images remains a difficult task, especially for high-throughput experiments where need for automation is strong. Low saliency of nuclei with respect to the background, variability of their intensity together with low signal-to-noise ratio in these images constitute a major challenge for mainstream algorithms of nuclei segmentation. In this work we introduce SalienceNet, an unsupervised deep learning-based method that uses the style transfer properties of cycleGAN to transform low saliency images into high saliency images, thus enabling accurate segmentation by downstream analysis methods, and that without need for any parameter tuning. We have acquired a novel dataset of organoid images with soSPIM, a microscopy technique that enables the acquisition of images in low-light conditions. Our experiments show that SalienceNet increased the saliency of these images up to the desired level. Moreover, we evaluated the impact of SalienceNet on segmentation for both Otsu thresholding and StarDist and have shown that enhancing nuclei with SalienceNet improved segmentation results using Otsu thresholding by 30% and using StarDist by 26% in terms of IOU when compared to segmentation of non-enhanced images. Together these results show that SalienceNet can be used as a common preprocessing step to automate nuclei segmentation pipelines for low-light microscopy images.

Light sheet fluorescence microscopy (LSFM) has become a leading standard in high-resolution imaging of living samples in 2- and 3-dimensions. Biological samples are however not restricted to a single observation plane and several molecular processes evolve rapidly in 3D. The conventional mechanical scanning required in LSFM limits the range of observable dynamics and are usually restricted in resolution. Here we introduce a new strategy for instantaneous volumetric excitation and volumetric imaging of single-molecules in cell aggregates. The technique combines, for the first time, the use of light sheet microscopy and multifocus microscopy (MFM) and enables a volumetric 4D imaging of biological samples with single-molecule resolution. We engineered the excitation beam to yield a modular and uniform excitation matching the observable detection range of MFM. The strength of the method is highlighted with examples of single-molecule 3D tracking and 3D super-resolution imaging in multicellular samples.

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimental and computational evidence that leukocytes do swim, and that efficient propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell external membrane. Our model consists of a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. Furthermore, continuous paddling is enabled by a combination of external treadmilling and selective recycling by internal vesicular transport of cortex-bound transmembrane proteins. This mechanism explains observations that swimming is five times slower than the retrograde flow of cortex and also that lymphocytes are motile in nonadherent confined environments. Resultantly, the ubiquitous ability of mammalian amoeboid cells to migrate in two dimensions or three dimensions and with or without adhesion can be explained for lymphocytes by a single machinery of heterogeneous membrane treadmilling.

Multicolor single-molecule localization microscopy (λSMLM) is a powerful technique to reveal the relative nanoscale organization and potential colocalization between different molecular species. While several standard analysis methods exist for pixel-based images, λSMLM still lacks such a standard. Moreover, existing methods only work on 2D data and are usually sensitive to the relative molecular organization, a very important parameter to consider in quantitative SMLM. Here, we present an efficient, parameter-free colocalization analysis method for 2D and 3D λSMLM using tessellation analysis. We demonstrate that our method allows for the efficient computation of several popular colocalization estimators directly from molecular coordinates and illustrate its capability to analyze multicolor SMLM data in a robust and efficient manner., Multicolour single-molecule localization microscopy lacks a standard analysis method. Here Levet et al. introduce Coloc-Tesseler, a parameter-free colocalisation analysis method based on tessellation analysis for the efficient analysis of multicolour SMLM data.

Single-molecule localization microscopy techniques have proven to be essential tools for quantitatively monitoring biological processes at unprecedented spatial resolution. However, these techniques are very low throughput and are not yet compatible with fully automated, multiparametric cellular assays. This shortcoming is primarily due to the huge amount of data generated during imaging and the lack of software for automation and dedicated data mining. We describe an automated quantitative single-molecule-based super-resolution methodology that operates in standard multiwell plates and uses analysis based on high-content screening and data-mining software. The workflow is compatible with fixed- and live-cell imaging and allows extraction of quantitative data like fluorophore photophysics, protein clustering or dynamic behavior of biomolecules. We demonstrate that the method is compatible with high-content screening using 3D dSTORM and DNA-PAINT based super-resolution microscopy as well as single-particle tracking.

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like fibroblasts crawling, relies on maturation of adhesion complexes and actin fiber traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimentally and computationally that leukocytes do swim, and that propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell envelope. We model the propulsion as a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. This mechanism explains that swimming is five times slower than the cortex retrograde flow. Resultantly the ubiquitous ability of mammalian amoeboid cells to migrate in various environments can be explained for lymphocytes by a single machinery of envelope treadmilling.

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this ‘autoblinking’ phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior of individual proteins in the plasma membrane as well as their molecular interaction with scaffold proteins at millisecond temporal resolution and high spatial resolution (

Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates. Using larger mirrors enabled us to broaden the capabilities of our system to image Drosophila embryos.

International audience; The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

International audience; The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

A Press release CEA, CNRS, UJF, INRA has been published for this publication (February 11th, 2013): http://www2.cnrs.fr/presse/communique/2987.htm; International audience; A promising approach to improve the performance of microelectronic devices is to build three-dimensional (3D) chips made of stacked circuits. However, a major hurdle lies in the fabrication of dense arrays of electrical interconnections between these layers, where accessibility is limited. Here we show that the directed growth and self-organization of actin filaments can offer a solution to this problem. We defined the shape and orientation of 3D actin networks through both micropatterning of actin nucleation factors and biochemical control of actin filament polymerization. Networks growing from two opposing layers were able to interpenetrate and form mechanically stable connections, which were then coated with gold using a selective metallization process. The electrical conductivity, robustness and modularity of the metallized self-organized connections make this approach potentially attractive for 3D chip manufacturing.

International audience; Alignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector; in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a 32 32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system. We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM).

International audience; We report a multi-confocal Fluorescence Correlation Spectroscopy (mFCS) technique that combines a Spatial Light Modulator (SLM), with an Electron Multiplying-CCD camera (EM-CCD). The SLM is used to produce a series of laser spots, while the pixels of the EM-CCD play the roles of virtual pinholes. The phase map addressed to the SLM, calculated by using the spherical wave approximation, makes it possible to produce several diffraction limited laser spots. The fastest acquisition mode leads to a time resolution of 100 microseconds. By using solutions of sulforhodamine G we demonstrated that the observation volumes are similar to that of a standard confocal set-up. mFCS experiments have also been conducted on two stable cell lines: mouse embryonic fibroblasts expressing eGFP-actin and H1299 cells expressing the heat shock factor fusion protein HSF1-eGFP. In the first case we could recover the diffusion constant of G-actin within the cytoplasm, although we were also sensitive to interactions with F-actin. Concerning HSF1, we could clearly observe the modifications of the number of molecules and of the HSF1 dynamics during heat shock.

Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules). Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCS-photobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand (@ 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other., Comment: 38 pages (avec les figures)

A novel portable fluorescence-based biosensor designed for field applications has been developed and successfully applied to the determination of two analytes, a neuropeptide model Substance P, and a small toxin Aflatoxin BI. A competitive immunoassay format called SPIT-FRI for Solid-Phase Immobilized Tripod for Fluorescent Renewable Immunoassay is used. The simplicity and robustness of the system apparatus is shown. Results clearly illustrate the assets of the developed array biosensor, by allowing in a portable instrument (i) simple regeneration steps, (ii) real-time monitoring, and (iii) multiplex detection thanks to a localized and cumulative fluorescence measurement.

A novel fluorescence-based array biosensor targeted for field applications, such as environmental monitoring, has been developed, and successfully applied to DNA hybridization assays. The purpose was to meet the demand for automated, portable but easy-to-maintain systems allowing continuous flow monitoring of surface reactions. The biosensor presented here can be distinguished from the existing systems by the optical method used, which provides an enhanced simplicity and robustness, and enables a simple maintenance by potentially unskilled personnel. The system is based on a conventional microscope slide which acts both as transducer and biological array sensor. The excited fluorescence is guided by total internal reflection into the slide to the detector which is directly interfaced to the slide. Each region of the sensor array is successively optically interrogated, and the detection of the corresponding fluorescent emission synchronized. A real-time three-analyte analysis is thus feasible without any mechanical scanning movement or optical imaging systems as generally used in the existing instruments. The ability of the biosensor to operate in continuous flow for several tens of hours has been demonstrated. The biosensor has been assessed in terms of stability, and slide-to-slide reproducibility, which is found to be less than 3.7%, thus far below the standard biological reproducibility. DNA hybridization assays were performed to estimate a limit of detection, which was found to be 16 mol/μm 2 , and to determine the reaction kinetics associated to the DNA model used. The developed biosensor is thus shown to be able to predict reaction kinetics, and to monitor in real time surface reactions between targets and probes.

Advances in scientific knowledge are often correlated with the development of new instrumental techniques. Biology is no exception to the rule. The recent development of super-resolution fluorescence microscopy techniques, awarded by the Nobel Prize in Chemistry in 2014, has revolutionized the way biological samples are observed by allowing to overcome the resolution limit dictated by light diffraction (~200 nm) and previously considered insurmountable. Among these techniques, Single Molecule Localization Microscopy (SMLM) allows to image the organization of proteins inside cells with the best spatial resolutions (~10 nm) and gives the possibility to follow their dynamics over time. These new capabilities open the way to a better understanding of the cellular machinery, essential to better fight against its dysfunctions at the origin of many diseases.However, the detection of single molecules for super-resolution imaging by SMLM is particularly challenging and requires both high optical sectioning and high signal collection efficiency. As a result, it is performed using specialized microscopy systems with extremely shallow sample penetration depths. The single-lens light sheet microscopy system developed in our team, named soSPIM, allows to overcome this limitation. By combining a deep optical sectioning with the use of a high numerical aperture objective, it allows the detection of individual molecules several tens of microns above a coverslip.Imaging whole volumes, such as whole cells, at these depths, however, rises new challenges. In excitation, light diffraction imposes a significant trade-off between the fineness of sectioning that can be achieved and the field of view. To overcome this, I sought to implement non-diffractive light sheet illumination to the soSPIM system. In detection, the performance of SMLM imaging depends directly on the optical quality of the system. Defects, called optical aberrations, appear systematically with the imaging depth. To correct these aberrations, I have implemented an adaptive optics system in the soSPIM microscope to allow precise 3D localization of individual molecules at depth. Finally, beyond the optical challenges, the acquisition and reconstruction procedures themselves present many difficulties. I have therefore developed several tools to consider spatial drifts and acquisition automation, and to reconstruct imaged volumes (~20x20x20 µm3) with nanometric resolutions. These developments have allowed the imaging of whole cells at 30 µm depth with resolutions of 5 nm radially (x, y) and 25 nm axially (z). This work provides a solid proof of concept of the soSPIM system's in depth SMLM imaging capability and paves the way for the observation of new biological structures at previously inaccessible nanometer resolutions., Les avancées des connaissances scientifiques sont souvent corrélées au développement de nouvelles techniques instrumentales. La biologie ne déroge pas la règle. Le développement récent des techniques de microscopie à fluorescence de super-résolution, récompensé par le prix Nobel de chimie en 2014, a révolutionné la façon d’observer les échantillons biologiques en permettant de dépasser la limite de résolution dictée par la diffraction de la lumière (~200 nm) et considérée jusqu’alors comme insurmontable. Parmi ces techniques, la microscopie par localisation de molécules individuelles (SMLM) permet d’imager l’organisation de protéines à l’intérieur des cellules avec les meilleures résolutions spatiales (~ 10 nm) et donne la possibilité de suivre leur dynamique au cours du temps. Ces nouvelles capacités ouvrent la voie à une meilleure compréhension de la machinerie cellulaire, essentielle pour mieux lutter contre ses dysfonctionnements, à l’origine de nombreuses maladies.La détection de molécules individuelles pour l’imagerie de super-résolution par SMLM est particulièrement difficile. Elle nécessite à la fois un sectionnement optique important et une grande efficacité dans la collecte du signal de fluorescence. En conséquence, elle est réalisée à l’aide de systèmes de microscopie spécialisés et dont la profondeur de pénétration dans les échantillons est extrêmement réduite. Le système de microscopie à feuille de lumière mono-objectif développé au sein de notre équipe, nommé soSPIM, permet de dépasser cette limitation. En combinant un sectionnement optique en profondeur avec l'utilisation d’un objectif à forte ouverture numérique, il permet la détection de molécules individuelles plusieurs dizaines de micromètres au-dessus d’une lamelle.L’imagerie de volumes entiers, tels que des cellules complètes, à ces profondeurs pose cependant d’autres défis. En excitation, la diffraction de la lumière impose un compromis important entre la finesse du sectionnement qu’il est possible de réaliser et le champ de vue. Pour tenter de dépasser cela, j’ai cherché à implémenter au système soSPIM une illumination par feuille de lumière non diffractive. En détection, les performances de l’imagerie par SMLM dépendent directement de la qualité optique du système. Or des défauts appelés aberrations optiques apparaissent systématiquement avec la profondeur d’imagerie. Pour corriger ces aberrations, j’ai implémenté un système d’optique adaptative au microscope soSPIM afin de permettre une localisation précise et en 3D de molécules individuelles en profondeur. Enfin, au-delà des défis optiques, les procédures d’acquisition et de reconstruction présentent elles-mêmes de nombreuses difficultés. J’ai donc mis en place plusieurs outils permettant de corriger les dérives spatiales, d’automatiser les acquisitions et de reconstruire les volumes imagés (~20x20x20 µm3) avec des résolutions nanométriques. Ces développements ont permis l’imagerie de cellules entières à 30 µm de profondeur avec des résolutions de 5 nm radialement (x,y) et 25 nm axialement (z). Ils forment ainsi une preuve de concept solide de la capacité du système soSPIM à l’imagerie par SMLM en profondeur. Ils ouvrent la voie à l’observation de nouvelles structures biologiques à des résolutions nanométriques jusqu’à présent inaccessibles.