Flexible implementation of modulated localisation microscopy based on DMD.

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.

Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets.

Fibroblasts generate topographical cues that steer cancer cell migration.

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells' migration through rearranging the extracellular matrix, secreting soluble factors, and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as a guidance cue for cancer cell migration.

Spatial organization of lysosomal exocytosis relies on membrane tension gradients.

Lysosomal exocytosis is involved in many key cellular processes but its spatiotemporal regulation is poorly known. Using total internal reflection fluorescence microscopy (TIRFM) and spatial statistics, we observed that lysosomal exocytosis is not random at the adhesive part of the plasma membrane of RPE1 cells but clustered at different scales. Although the rate of exocytosis is regulated by the actin cytoskeleton, neither interfering with actin or microtubule dynamics by drug treatments alters its spatial organization.

Time-modulated excitation for enhanced single-molecule localization microscopy.

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.

Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions.

Focal adhesions (FAs) initiate chemical and mechanical signals involved in cell polarity, migration, proliferation and differentiation. Super-resolution microscopy revealed that FAs are organized at the nanoscale into functional layers from the lower plasma membrane to the upper actin cytoskeleton. Yet, how FAs proteins are guided into specific nano-layers to promote interaction with given targets is unknown.

Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields.

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination.

Combining 3D single molecule localization strategies for reproducible bioimaging.

Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 μm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range.

Supercritical angle fluorescence for enhanced axial sectioning in STED microscopy.

We demonstrate subwavelength axial sectioning on biological samples with a stimulated emission depletion (STED) microscope combined with supercritical angle fluorescence (SAF) detection. SAF imaging is a powerful technique for imaging the membrane of the cell based on the direct exploitation of the fluorophore emission properties. Indeed, only when fluorophores are close to the interface can their evanescent near-field emission become propagative and be detected beyond the critical angle.

Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.

Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical, , and animal studies report treatment failure, notably against The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity against varies significantly with the growth state and strain, according to the membrane fatty acid composition.

Aberration-accounting calibration for 3D single-molecule localization microscopy.

We propose a straightforward sample-based technique to calibrate the axial detection in 3D single-molecule localization microscopy. Using microspheres coated with fluorescent molecules, the calibration curves of point spread function-shaping or intensity-based measurements can be obtained over the imaging depth range. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.

Podosome Force Generation Machinery: A Local Balance between Protrusion at the Core and Traction at the Ring.

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields.

Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation.

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive.

Label-free evanescent microscopy for membrane nano-tomography in living cells.

We show that through-the-objective evanescent microscopy (epi-EM) is a powerful technique to image membranes in living cells. Readily implementable on a standard inverted microscope, this technique enables full-field and real-time tracking of membrane processes without labeling and thus signal fading. In addition, we demonstrate that the membrane/interface distance can be retrieved with 10 nm precision using a multilayer Fresnel model.

Fast label-free cytoskeletal network imaging in living mammalian cells.

We present a full-field technique that allows label-free cytoskeletal network imaging inside living cells. This noninvasive technique allows monitoring of the cytoskeleton dynamics as well as interactions between the latter and organelles on any timescale. It is based on high-resolution quantitative phase imaging (modified Quadriwave lateral shearing interferometry) and can be directly implemented using any optical microscope without modification.

Confocal supercritical angle microscopy for cell membrane imaging.

We demonstrate subwavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fluorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modification on the detection channel.

Full-field near-field optical microscope for cell imaging.

We report a new full-field fluorescence microscopy method for imaging live cell membranes based on supercritical near-field emission. This technique consists of extracting the self-interference between undercritical and supercritical light by simple image subtraction. In the objective back focal plane, this is equivalent to adding a virtual mask blocking the subcritical emission.

Homodimerization of amyloid precursor protein at the plasma membrane: a homoFRET study by time-resolved fluorescence anisotropy imaging.

Classical FRET (Förster Resonance Energy Transfer) using two fluorescent labels (one for the donor and another one for the acceptor) is not efficient for studying the homodimerization of a protein as only half of the homodimers formed can be identified by this technique. We thus resorted to homoFRET detected by time-resolved Fluorescence Anisotropy IMaging (tr-FAIM). To specifically image the plasma membrane of living cells, an original combination of tr-FAIM and Total Internal Reflection Fluorescence Lifetime Imaging Microscope (TIRFLIM) was implemented.

Simultaneous optically sectioned fluorescence and optical coherence microscopy with full-field illumination.

Full-field optical coherence microscopy (FF-OCM) and optically sectioned fluorescence microscopy are two imaging techniques that are implemented here in a novel dual modality instrument. The two imaging modalities use a broad field illumination to acquire the entire field of view without raster scanning. Optical sectioning is achieved in both imaging modalities owing to the coherence gating property of light for FF-OCM, and a structured illumination setup for fluorescence microscopy.

Determination of lipid raft partitioning of fluorescently-tagged probes in living cells by Fluorescence Correlation Spectroscopy (FCS).

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases.

Full-field supercritical angle fluorescence microscopy for live cell imaging.

We introduce a full-field fluorescence imaging technique with axial confinement of about 100 nm at the sample/substrate interface. Contrary to standard surface imaging techniques, this confinement is obtained through emission filtering. This technique is based on supercritical emission selectivity.

Local cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and rapid endocytosis.

Amyloid peptide (Aβ) is generated by sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Aβ production increases after plasma membrane cholesterol loading through unknown mechanisms. To determine how APP-Bace1 proximity affects this phenomenon, we developed a fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET) technique for visualization of these molecules either by epifluorescence or at the plasma membrane only using total internal reflection fluorescence.

Picosecond polarized supercontinuum generation controlled by intermodal four-wave mixing for fluorescence lifetime imaging microscopy.

We present the generation of a picosecond polarized supercontinuum in highly birefringent multimodal microstructured fiber. The initial steps of the spectral broadening are dominated by intermodal four-wave mixing controlled by the specific fiber design. Using a low repetition rate ultra-stable solid state laser, a pulse train well-suited for versatile time-domain fluorescence lifetime imaging applications is obtained.