High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation.

Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below the diffraction limit. Here we present enhanced super-resolution radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to the original SRRF method. eSRRF incorporates automated parameter optimization based on the data itself, giving insight into the trade-off between resolution and fidelity.

Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells.

Exerting forces on biomolecules inside living cells would allow us to probe their dynamic interactions in their native environment. Magnetic iron oxide nanoparticles represent a unique tool capable of pulling on biomolecules with the application of an external magnetic field gradient; however, their use has been restricted to biomolecules accessible from the extracellular medium. Targeting intracellular biomolecules represents an additional challenge due to potential nonspecific interactions with cytoplasmic or nuclear components.

A human septin octamer complex sensitive to membrane curvature drives membrane deformation with a specific mesh-like organization.

Septins are cytoskeletal proteins interacting with the inner plasma membrane and other cytoskeletal partners. Being key in membrane remodeling processes, they often localize at specific micrometric curvatures. To analyze the behavior of human septins at the membrane and decouple their role from other partners, we used a combination of bottom-up in vitro methods.

SPITFIR(e): a supermaneuverable algorithm for fast denoising and deconvolution of 3D fluorescence microscopy images and videos.

Modern fluorescent microscopy imaging is still limited by the optical aberrations and the photon budget available in the specimen. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when restraining the illumination to low levels in order to limit photo-damages. Here, we report SPITFIR(e) a flexible method designed to accurately and quickly restore 2D-3D fluorescence microscopy images and videos (4D images).

New Approach to Accelerated Image Annotation by Leveraging Virtual Reality and Cloud Computing.

Three-dimensional imaging is at the core of medical imaging and is becoming a standard in biological research. As a result, there is an increasing need to visualize, analyze and interact with data in a natural three-dimensional context. By combining stereoscopy and motion tracking, commercial virtual reality (VR) headsets provide a solution to this critical visualization challenge by allowing users to view volumetric image stacks in a highly intuitive fashion.

Towards Human in the Loop Analysis of Complex Point Clouds: Advanced Visualizations, Quantifications, and Communication Features in Virtual Reality.

Multiple fields in biological and medical research produce large amounts of point cloud data with high dimensionality and complexity. In addition, a large set of experiments generate point clouds, including segmented medical data or single-molecule localization microscopy. In the latter, individual molecules are observed within their natural cellular environment.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases.

Molecular basis of CTCF binding polarity in genome folding.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin.

Genuage: visualize and analyze multidimensional single-molecule point cloud data in virtual reality.

Experimentally recorded point cloud data, such as those generated by single-molecule localization microscopy, are continuously increasing in size and dimension. Gaining an intuitive understanding and facilitating the analysis of such multidimensional data remains challenging. Here we report a new open-source software platform, Genuage, that enables the easy perception of, interaction with and analysis of multidimensional point clouds in virtual reality.

DIVA: Natural Navigation Inside 3D Images Using Virtual Reality.

As three-dimensional microscopy becomes commonplace in biological research, there is an increasing need for researchers to be able to view experimental image stacks in a natural three-dimensional viewing context. Through stereoscopy and motion tracking, commercial virtual reality headsets provide a solution to this important visualization challenge by allowing researchers to view volumetric objects in an entirely intuitive fashion. With this motivation, we present DIVA, a user-friendly software tool that automatically creates detailed three-dimensional reconstructions of raw experimental image stacks that are integrated in virtual reality.

High-resolution visualization of H3 variants during replication reveals their controlled recycling.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.

Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings.

Multifocus microscopy (MFM) allows sensitive and fast three-dimensional imaging. It relies on the efficient design of diffraction phase gratings yielding homogeneous intensities in desired diffraction orders. Such performances are however guaranteed only for a specific wavelength.

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms.

PSF engineering in multifocus microscopy for increased depth volumetric imaging.

Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm.

Full determination of single ferroelectric nanocrystal orientation by Pockels electro-optic microscopy.

We present a nanoscale electro-optic imaging method allowing access to the phase response, which is not amenable to classical second-harmonic generation microscopy. This approach is used to infer the vectorial orientation of single domain ferroelectric nanocrystals, based on polarization-resolved Pockels microscopy. The electro-optic phase response of KTP nanoparticles yields the full orientation in the laboratory frame of randomly dispersed single nanoparticles, together with their electric polarization dipole.

Accessing the third dimension in localization-based super-resolution microscopy.

Only a few years after its inception, localization-based super-resolution microscopy has become widely employed in biological studies. Yet, it is primarily used in two-dimensional imaging and accessing the organization of cellular structures at the nanoscale in three dimensions (3D) still poses important challenges. Here, we review optical and computational techniques that enable the 3D localization of individual emitters and the reconstruction of 3D super-resolution images.

Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres.

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation.

Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4.

Real-time dynamics of RNA polymerase II clustering in live human cells.

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Phase resolved interferometric spectral modulation (PRISM) for ultrafast pulse measurement and compression.

We show through experiments and simulations that parallel phase modulation, a technique developed in the field of adaptive optics, can be employed to quickly determine the spectral phase profile of ultrafast laser pulses and to perform phase compensation as well as pulse shaping. Different from many existing ultrafast pulse measurement methods, the technique reported here requires no spectrum measurements of nonlinear signals. Instead, the power of nonlinear signals is used directly to quickly measure the spectral phase, a convenient feature for applications such as two-photon fluorescence microscopy.

Electro-optical Pockels scattering from a single nanocrystal.

The electro-optical Pockels response from a single non-centrosymmetric nanocrystal is reported. High sensitivity to the weak electric-field dependent nonlinear scattering is achieved through a dedicated imaging interferometric microscope and the linear dependence of electro-optical signal upon the applied field is checked. Using different incident light polarization states, a priori random spatial orientation of the crystal can be inferred.

Electro-optical imaging microscopy of dye-doped artificial lipidic membranes.

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane.