Rémi Galland

Reprogramming cell shape with laser nano-patterning

Cell shape in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the cell microenvironment. Here, we manipulate the shape of living cells in real time by using a tightly focused pulsed laser to introduce additional geometrically defined adhesion sites. The sub-micrometer resolution of the laser patterning allowed us to identify the critical distances between cell adhesion sites required for cell shape extension and contraction. This easy-to-handle method allows the precise control of specific actin-based structures that regulate cell architecture. Actin filament bundles or branched meshworks were induced, displaced or removed in response to specific dynamic modifications of the cell adhesion pattern. Isotropic branched actin meshworks could be forced to assemble new stress fibers locally and polarised in response to specific geometrical cues.

3D high- and super-resolution imaging using single-objective SPIM.

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.

Fabrication of three-dimensional electrical connections by means of directed actin self-organization

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.

A novel fluorescence-based array biosensor : Principle and application to DNA hybridization assays

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/microm(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.

Automatic laser alignment for multifocal microscopy using a LCOS SLM and a 32×32 pixel CMOS SPAD array

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).

Localization-based super-resolution imaging meets high-content screening

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.

3D Protein Dynamics in the Cell Nucleus

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus. We are able to acquire >3400 autocorrelation functions at multiple spatial positions within a nucleus, without significant photobleaching, allowing us to make reliable estimates of diffusion dynamics. Using this tool, we demonstrate spatial heterogeneity in Polymerase II dynamics in live U2OS cells. Further, we provide detailed measurements of human-Yes-associated protein diffusion dynamics in a human gastric cancer epithelial cell line.

Directed Actin Assembly and Motility

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.

Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

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 (approximately = 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. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its nonradiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

A Robust Fluorescence Multiplex Immunoassay Biosensor Designed for Field Applications

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.