BMP2 Binds Non-Specifically to PEG-Passivated Biomaterials and Induces pSMAD 1/5/9 Signalling.

Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore how extracellular matrix components influence cellular response. In this study, the previously developed streptavidin platforms is aimed to use to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, it is observed that the interpretation of findings is skewed due to the GAG-unrelated, non-specific binding of BMP2 on components of biomaterials.

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells.

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD).

Automated Fabrication of Streptavidin-Based Self-assembled Materials for High-Content Analysis of Cellular Response to Growth Factors.

The automation of liquid-handling routines offers great potential for fast, reproducible, and labor-reduced biomaterial fabrication but also requires the development of special protocols. Competitive systems demand for a high degree in miniaturization and parallelization while maintaining flexibility regarding the experimental design. Today, there are only a few possibilities for automated fabrication of biomaterials inside multiwell plates.

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems.

Quantitative analysis of hidden particles diffusing behind a scattering layer using speckle correlation.

Speckle-correlation imaging is a family of methods that makes use of the "memory effect" to image objects hidden behind visually opaque layers. Here, we show that a correlation analysis can be applied to quantitative imaging of an ensemble of dynamic fluorescent beads diffusing on a 2D surface. We use an epi-fluorescence microscope where both the illumination and detection light patterns are speckled, due to light scattering by a thin disordered layer.

Measuring cell displacements in opaque tissues: dynamic light scattering in the multiple scattering regime.

Coherent light scattered by tissues brings structural and dynamic information, at depth, that standard imaging techniques cannot reach. Dynamics of cells or sub-cellular elements can be measured thanks to dynamic light scattering in thin samples (single scattering regime) or thanks to diffusive wave spectroscopy in thick samples (diffusion regime). Here, we address the intermediate regime and provide an analytical relationship between scattered light fluctuations and the distribution of cell displacements as a function of time.

Confocal fluorescence correlation spectroscopy through a sparse layer of scattering objects.

In the presence of strong light scattering, as often encountered in biological tissue, optical microscopy becomes challenging and technical demanding. Beside image quality, the quantitative determination of molecular properties is also strongly affected by scattering. We have carried out fluorescence correlation spectroscopy (FCS) experiments, in a solution of fluorophores, through a sparse scattering layer made of dielectric beads.

Cellular tension encodes local Src-dependent differential β and β integrin mobility.

Integrins are transmembrane receptors that have a pivotal role in mechanotransduction processes by connecting the extracellular matrix to the cytoskeleton. Although it is well established that integrin activation/inhibition cycles are due to highly dynamic interactions, whether integrin mobility depends on local tension and cytoskeletal organization remains surprisingly unclear. Using an original approach combining micropatterning on glass substrates to induce standardized local mechanical constraints within a single cell with temporal image correlation spectroscopy, we measured the mechanosensitive response of integrin mobility at the whole cell level and in adhesion sites under different mechanical constraints.

Optimizing the metric in sensorless adaptive optical microscopy with fluorescence fluctuations.

Adaptive optics (AO) strategies using optimization-based, sensorless approaches are widely used, especially for microscopy applications. To converge rapidly to the best correction, such approaches require that a quality metric and a set of modes be chosen optimally. Fluorescence fluctuations microscopy, a family of methods that provides quantitative measurements of molecular concentration and mobility in living specimen, is in particular need of adaptive optics, since its results can be strongly biased by optical aberrations.

Data on blueberry peroxidase kinetic characterization and stability towards thermal and high pressure processing.

The data presented in this article are related to a research article entitled 'Thermal and high pressure inactivation kinetics of blueberry peroxidase' (Terefe et al., 2017) [1]. In this article, we report original data on the activity of partially purified blueberry peroxidase at different concentrations of hydrogen peroxide and phenlylenediamine as substrates and the effects of thermal and high pressure processing on the activity of the enzyme.

Thermal and high pressure inactivation kinetics of blueberry peroxidase.

This study for the first time investigated the stability and inactivation kinetics of blueberry peroxidase in model systems (McIlvaine buffer, pH=3.6, the typical pH of blueberry juice) during thermal (40-80°C) and combined high pressure-thermal processing (0.1-690MPa, 30-90°C).

αvβ3 integrins negatively regulate cellular forces by phosphorylation of its distal NPXY site.

Background Information: Integrins are key receptors that allow cells to sense and respond to their mechanical environment. Although they bind the same ligand, β1 and β3 integrins have distinct and cooperative roles in mechanotransduction.

Results: Using traction force microscopy on unconstrained cells, we show that deleting β3 causes traction forces to increase, whereas the deletion of β1 integrin results in a strong decrease of contractile forces.

Effect of an osmotic stress on multicellular aggregates.

There is increasing evidence that multicellular structures respond to mechanical cues, such as the confinement and compression exerted by the surrounding environment. In order to understand the response of tissues to stress, we investigate the effect of an isotropic stress on different biological systems. The stress is generated using the osmotic pressure induced by a biocompatible polymer.

Blueberry polyphenol oxidase: Characterization and the kinetics of thermal and high pressure activation and inactivation.

Partially purified blueberry polyphenol oxidase (PPO) in Mcllvaine buffer (pH=3.6, typical pH of blueberry juice) was subjected to processing at isothermal-isobaric conditions at temperatures from 30 to 80 °C and pressure from 0.1 to 700 MPa.

Fluorescent correlation spectroscopy measurements with adaptive optics in the intercellular space of spheroids.

In this study we demonstrate the use of adaptive optics to correct the biasing effects of optical aberrations when measuring the dynamics of molecules diffusing between cells in multicellular spheroids. Our results indicate that, on average, adaptive optics leads to a reduction of the 3D size of the point spread function that is statistically significant in terms of measured number of molecules and diffusion time. The sensorless approach, which uses the molecular brightness as optimization metric, is validated in a complex, highly heterogeneous, biological environment.

Correction of cell-induced optical aberrations in a fluorescence fluctuation microscope.

We describe the effect of optical aberrations on fluorescence fluctuations microscopy (FFM), when focusing through a single living cell. FFM measurements are performed in an aqueous fluorescent solution and prove to be a highly sensitive tool to assess the optical aberrations introduced by the cell. We demonstrate an adaptive optics (AO) system to remove the aberration-related bias in the FFM measurements.

Dynamics of the full length and mutated heat shock factor 1 in human cells.

Heat shock factor 1 is the key transcription factor of the heat shock response. Its function is to protect the cell against the deleterious effects of stress. Upon stress, HSF1 binds to and transcribes hsp genes and repeated satellite III (sat III) sequences present at the 9q12 locus.

Cellular response to heat shock studied by multiconfocal fluorescence correlation spectroscopy.

Heat shock triggers a transient and ubiquitous response, the function of which is to protect cells against stress-induced damage. The heat-shock response is controlled by a key transcription factor known as heat shock factor 1 (HSF1). We have developed a multiconfocal fluorescence correlation spectroscopy setup to measure the dynamics of HSF1 during the course of the heat-shock response.

Adaptive optics for fluorescence correlation spectroscopy.

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. In this paper, we quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics system for aberration correction.

Multi-confocal fluorescence correlation spectroscopy.

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.

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.

Spatial fluorescence cross-correlation spectroscopy by means of a spatial light modulator.

Spatial fluorescence cross-correlation spectroscopy is a rarely investigated version of fluorescence correlation spectroscopy, in which the fluorescence signals from different observation volumes are cross-correlated. In the reported experiments, two observation volumes, typically shifted by a few microm, are produced, with a spatial light modulator and two adjustable pinholes. We illustrated the feasibility and potentiality of this technique by: i) measuring molecular flows, in the range 0.

Spatial fluorescence cross-correlation spectroscopy.

We present an alternative method for diffusion measurements of fluorescent species in solution by use of confocal microscopy and fluorescence correlation spectroscopy techniques. It consists of making a time and spatial dual correlation in which one detects the fluorescence signals from two nearby separate confocal volumes and cross correlates them. To improve the spatial discrimination between the two confocal volumes we propose filtering of fluorescence photocounts by rejecting the fluorescence background, which corresponds to particles located far from the center of the detection volumes.