Aberration-free 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 (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.Comment: 8 pages, 2 figures. Submitted to Optics Letters on October 12th, 201

Latest trends in bioimaging and building a proactive network of early-career young scientists around bioimaging in Europe

Biological research is in constant need of new methodological developments to assess organization and functions at various scales ranging from whole organisms to interactions between proteins. One of the main ways to evidence and quantify biological phenomena is imaging. Fluorescence microscopy and label-free microscopy are in particular highly active fields of research due to their compatibility with living samples as well as their versatility. The Imabio Young Scientists Network (YSN) is a group of young scientists (PhD students, postdocs and engineers) who are excited about bioimaging and aim to create a proactive network of researchers with the same interest. YSN is endorsed by the bioimaging network GDR Imabio in France, where the initiative was started in 2019. Since then, we aim to organize the Imabio YSN conference every year to expand the network to other European countries, establish new collaborations and ignite new scientific ideas. From 6-8 July 2022, the YSN including researchers from the domains of life sciences, chemistry, physics and computational sciences met at the Third Imabio YSN Conference 2022 in Lyon to discuss the latest bioimaging technologies and biological discoveries. In this Meeting Review, we describe the essence of the scientific debates, highlight remarkable talks, and focus on the Career Development session, which is unique to the YSN conference, providing a career perspective to young scientists and help to answer all their questions at this career stage. This conference was a truly interdisciplinary reunion of scientists who are eager to push the frontiers of bioimaging in order to understand the complexity of biological systems

Additive Manufacturing of 3D Luminescent ZrO2:Eu3+ Architectures

Implementation of more refined structures at the nano to microscale is expected to advance applications in optics and photonics. This work presents the additive manufacturing of 3D luminescent microarchitectures emitting light in the visible range. A tailor-made organo-metallic resin suitable for two-photon lithography is developed, which upon thermal treatment in an oxygen-rich atmosphere allows the creation of silicon-free tetragonal (t-) and monoclinic (m-) ZrO2. The approach is unique because the tailor-made Zr-resin is different from what is achieved in other reported approaches based on sol−gel resins. The Zr-resin is compatible with the Eu-rich dopant, a luminescent activator, which enables to tune the optical properties of the ZrO2 structures upon annealing. The emission characteristics of the Eu-doped ZrO2 microstructures are investigated in detail with cathodoluminescence and compared with the intrinsic optical properties of the ZrO2. The hosted Eu has an orange−red emission showcased using fluorescence microscopy. The presented structuring technology provides a new platform for the future development of 3D luminescent devices

Single-molecule super-resolution imaging of microfabricated 3D substrates for 3D cell culture (Conference Presentation)

The potential of micro-and nanofabricated samples as a platform to modulate cell behavior using 3D physical cues has shown tremendous interest in cell differentiation. Investigating cell behavior and organization at the molecular level requires advanced imaging techniques. We produced multiscale 3D substrates with glass fractal pyramids composed of several generations of octahedra of decreasing sizes, which allow direct observation of cells in 3D SMLM. We show how these samples can be fluorescently labeled and used as a self-referenced sample for calibration and resolution measurements. Moreover, we perform quantitative 3D SMLM on cells growing on such fractal substrates, observing spheroid-like behavior

Additive Manufacturing of 3D Luminescent ZrO2:Eu3+ Architectures

European Research Council (ERC) under the Horizon 2020 research and innovation programme of the European Union. Grant Numbers: 742004, 949626; University of California Institute for Mexico and the United States. Grant Number: CN19137; CONACYT. Grant Number: 284667; University of Twent

3D topographies promote macrophage M2d-Subset differentiation

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments

Combining 3D single molecule localization strategies for reproducible bioimaging

International audienceHere, 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. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-μm range by direct merging of multiple acquisitions at different depths

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

International audienceDaptomycin 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, in vitro, and animal studies report treatment failure, notably against Staphylococcus aureus. 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 S. aureus varies significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treat S. aureus is not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies

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. Using single protein tracking, super-resolution microscopy and functional assays, we link the molecular behavior and 3D nanoscale localization of kindlin with its function in integrin activation inside FAs. We show that immobilization of integrins in FAs depends on interaction with kindlin. Unlike talin, kindlin displays free diffusion along the plasma membrane outside and inside FAs. We demonstrate that the kindlin Pleckstrin Homology domain promotes membrane diffusion and localization to the membrane-proximal integrin nano-layer, necessary for kindlin enrichment and function in FAs. Using kindlin-deficient cells, we show that kindlin membrane localization and diffusion are crucial for integrin activation, cell spreading and FAs formation. Thus, kindlin uses a different route than talin to reach and activate integrins, providing a possible molecular basis for their complementarity during integrin activation