Fluorescence to measure light intensity.

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.

An actin-based viscoplastic lock ensures progressive body-axis elongation.

Body-axis elongation constitutes a key step in animal development, laying out the final form of the entire animal. It relies on the interplay between intrinsic forces generated by molecular motors1-3, extrinsic forces exerted by adjacent cells4-7 and mechanical resistance forces due to tissue elasticity or friction8-10. Understanding how mechanical forces influence morphogenesis at the cellular and molecular level remains a challenge1. Recent work has outlined how small incremental steps power cell-autonomous epithelial shape changes1-3, which suggests the existence of specific mechanisms that stabilize cell shapes and counteract cell elasticity. Beyond the twofold stage, embryonic elongation in Caenorhabditis elegans is dependent on both muscle activity7 and the epidermis; the tension generated by muscle activity triggers a mechanotransduction pathway in the epidermis that promotes axis elongation7. Here we identify a network that stabilizes cell shapes in C. elegans embryos at a stage that involves non-autonomous mechanical interactions between epithelia and contractile cells. We searched for factors genetically or molecularly interacting with the p21-activating kinase homologue PAK-1 and acting in this pathway, thereby identifying the α-spectrin SPC-1. Combined absence of PAK-1 and SPC-1 induced complete axis retraction, owing to defective epidermal actin stress fibre. Modelling predicts that a mechanical viscoplastic deformation process can account for embryo shape stabilization. Molecular analysis suggests that the cellular basis for viscoplasticity originates from progressive shortening of epidermal microfilaments that are induced by muscle contractions relayed by actin-severing proteins and from formin homology 2 domain-containing protein 1 (FHOD-1) formin bundling. Our work thus identifies an essential molecular lock acting in a developmental ratchet-like process.

Publisher Correction: An actin-based viscoplastic lock ensures progressive body-axis elongation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: An actin-based viscoplastic lock ensures progressive body-axis elongation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The TPLATE complex mediates membrane bending during plant clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.

Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae.

Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment.

Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging.

Using the haloarchaeon Haloferax volcanii as a model, we developed nascent DNA labeling and the functional GFP-labeled single-stranded binding protein RPA2 as novel tools to gain new insight into DNA replication and repair in live haloarchaeal cells. Our quantitative fluorescence microscopy data revealed that RPA2 forms distinct replication structures that dynamically responded to replication stress and DNA damaging agents. The number of the RPA2 foci per cell followed a probabilistic Poisson distribution, implying hitherto unnoticed stochastic cell-to-cell variation in haloarchaeal DNA replication and repair processes. The size range of haloarchaeal replication structures is very similar to those observed earlier in eukaryotic cells. The improved lateral resolution of 3D-SIM fluorescence microscopy allowed proposing that inhibition of DNA synthesis results in localized replication foci clustering and facilitated observation of RPA2 complexes brought about by chemical agents creating DNA double-strand breaks. Altogether our in vivo observations are compatible with earlier in vitro studies on archaeal single-stranded DNA binding proteins. Our work thus underlines the great potential of live cell imaging for unraveling the dynamic nature of transient molecular interactions that underpin fundamental molecular processes in the Third domain of life.

Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons.

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes.

In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near-infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850-1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post-acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes.

Proton Transfer and Structure-Specific Fluorescence in Hydrogen Bond-Rich Protein Structures.

Protein structures which form fibrils have recently been shown to absorb light at energies in the near UV range and to exhibit a structure-specific fluorescence in the visible range even in the absence of aromatic amino acids. However, the molecular origin of this phenomenon has so far remained elusive. Here, we combine ab initio molecular dynamics simulations and fluorescence spectroscopy to demonstrate that these intrinsically fluorescent protein fibrils are permissive to proton transfer across hydrogen bonds which can lower electron excitation energies and thereby decrease the likelihood of energy dissipation associated with conventional hydrogen bonds. The importance of proton transfer on the intrinsic fluorescence observed in protein fibrils is signified by large reductions in the fluorescence intensity upon either fully protonating, or deprotonating, the fibrils at pH = 0 or 14, respectively. Thus, our results point to the existence of a structure-specific fluorophore that does not require the presence of aromatic residues or multiple bond conjugation that characterize conventional fluorescent systems. The phenomenon may have a wide range of implications in biological systems and in the design of self-assembled functional materials.

Multicolor two-photon tissue imaging by wavelength mixing.

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

In Situ Visualization of Block Copolymer Self-Assembly in Organic Media by Super-Resolution Fluorescence Microscopy.

Analytical methods that enable visualization of nanomaterials derived from solution self-assembly processes in organic solvents are highly desirable. Herein, we demonstrate the use of stimulated emission depletion microscopy (STED) and single molecule localization microscopy (SMLM) to map living crystallization-driven block copolymer (BCP) self-assembly in organic media at the sub-diffraction scale. Four different dyes were successfully used for single-colour super-resolution imaging of the BCP nanostructures allowing micelle length distributions to be determined in situ. Dual-colour SMLM imaging was used to measure and compare the rate of addition of red fluorescent BCP to the termini of green fluorescent seed micelles to generate block comicelles. Although well-established for aqueous systems, the results highlight the potential of super-resolution microscopy techniques for the interrogation of self-assembly processes in organic media.

Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF.

Spatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF).

Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy.

A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1-3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500-600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior-superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.

Third harmonic imaging contrast from tubular structures in the presence of index discontinuity.

Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2[Formula: see text]-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarization-resolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries.

Chromatically Corrected Multicolor Multiphoton Microscopy.

Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this light-efficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging.

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy.

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy.

Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression.

The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA-negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaAneg daughter, which regains the size and behavior of its mother, and a neurogenic deltaApos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaAneg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations.