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1.
Cell ; 156(6): 1274-1285, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24630727

RESUMO

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. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.


Assuntos
DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única , Animais , Imunoprecipitação da Cromatina , Epigênese Genética , Estudo de Associação Genômica Ampla , Cinética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética
2.
Nat Methods ; 20(12): 1949-1956, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37957430

RESUMO

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. We demonstrate eSRRF across a range of imaging modalities and biological systems. Notably, we extend eSRRF to three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric super-resolution imaging with an acquisition speed of ~1 volume per second. eSRRF provides an accessible super-resolution approach, maximizing information extraction across varied experimental conditions while minimizing artifacts. Its optimal parameter prediction strategy is generalizable, moving toward unbiased and optimized analyses in super-resolution microscopy.


Assuntos
Artefatos , Microscopia de Fluorescência/métodos
3.
J Cell Sci ; 136(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37305997

RESUMO

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. We assayed their ultrastructural organization, their curvature sensitivity, as well as their role in membrane reshaping. On membranes, human septins organize into a two-layered mesh of orthogonal filaments, instead of generating parallel sheets of filaments observed for budding yeast septins. This peculiar mesh organization is sensitive to micrometric curvature and drives membrane reshaping as well. The observed membrane deformations together with the filamentous organization are recapitulated in a coarse-grained computed simulation to understand their mechanisms. Our results highlight the specific organization and behavior of animal septins at the membrane as opposed to those of fungal proteins.


Assuntos
Citoesqueleto , Septinas , Animais , Humanos , Septinas/genética , Membranas , Membrana Celular , Bioensaio
4.
Nano Lett ; 23(13): 5919-5926, 2023 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-37390368

RESUMO

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. We present the synthesis of sulfobetaine-phosphonate block copolymer ligands, which provide magnetic nanoparticles that are stealthy and targetable in living cells. We demonstrate, for the first time, their efficient targeting in the nucleus and their use for magnetic micromanipulation of a specific genomic locus in living cells. We believe that these stable and sensitive magnetic nanoprobes represent a promising tool to manipulate specific biomolecules in living cells and probe the mechanical properties of living matter at the molecular scale.


Assuntos
Nanopartículas , Polímeros , Micromanipulação , Genômica , Nanopartículas Magnéticas de Óxido de Ferro , Fenômenos Magnéticos
5.
Nat Methods ; 17(11): 1100-1102, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32958921

RESUMO

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. Genuage is compatible with arbitrary multidimensional data extending beyond single-molecule localization microscopy.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imagem Individual de Molécula/métodos , Software , Realidade Virtual , Algoritmos , Inteligência Artificial , Células HeLa , Humanos , Mitocôndrias/química , Tubulina (Proteína)/química
6.
Proc Natl Acad Sci U S A ; 111(49): 17480-5, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422417

RESUMO

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼ 4-µm-deep volume, with lateral and axial localization precisions of ∼ 20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.


Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Calibragem , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Modelos Moleculares , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo
7.
Nat Methods ; 10(1): 60-3, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23223154

RESUMO

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.


Assuntos
Caenorhabditis elegans/citologia , Rastreamento de Células , Imageamento Tridimensional/métodos , Microscopia de Fluorescência , Neurônios/citologia , Saccharomyces cerevisiae/citologia , Animais , Neoplasias Ósseas/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Osteossarcoma/enzimologia , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Appl Opt ; 54(11): 3412-21, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25967332

RESUMO

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. The complete vector determination of the dipole orientation is a prerequisite to important applications including ferroelectric nanodomain orientation, membrane potential imaging, and rotational dynamics of single biomolecules.

9.
Phys Chem Chem Phys ; 16(31): 16340-8, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-24901106

RESUMO

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. These techniques are grouped into three main categories: PSF engineering, multiple plane imaging and interferometric approaches. We provide an overview of their technical implementation as well as commentary on their applicability. Finally, we discuss future trends in 3D localization-based super-resolution microscopy.


Assuntos
Microscopia/métodos , Conformação Molecular , Algoritmos
10.
Sci Rep ; 13(1): 1489, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707688

RESUMO

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). We designed a generic sparse-promoting regularizer to subtract undesirable out-of-focus background and we developed a primal-dual algorithm for fast optimization. SPITFIR(e) is a "swiss-knife" method for practitioners as it adapts to any microscopy techniques, to various sources of signal degradation (noise, blur), to variable image contents, as well as to low signal-to-noise ratios. Our method outperforms existing state-of-the-art algorithms, and is more flexible than supervised deep-learning methods requiring ground truth datasets. The performance, the flexibility, and the ability to push the spatiotemporal resolution limit of sub-diffracted fluorescence microscopy techniques are demonstrated on experimental datasets acquired with various microscopy techniques from 3D spinning-disk confocal up to lattice light sheet microscopy.

11.
J Vis Exp ; (186)2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-36063014

RESUMO

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. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.


Assuntos
Septinas , Lipossomas Unilamelares , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Bicamadas Lipídicas/química , Mamíferos/metabolismo , Saccharomyces cerevisiae/metabolismo , Septinas/química , Septinas/genética , Septinas/metabolismo , Lipossomas Unilamelares/metabolismo
12.
Front Bioinform ; 2: 997082, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36304296

RESUMO

Microscopy image observation is commonly performed on 2D screens, which limits human capacities to grasp volumetric, complex, and discrete biological dynamics. With the massive production of multidimensional images (3D + time, multi-channels) and derived images (e.g., restored images, segmentation maps, and object tracks), scientists need appropriate visualization and navigation methods to better apprehend the amount of information in their content. New modes of visualization have emerged, including virtual reality (VR)/augmented reality (AR) approaches which should allow more accurate analysis and exploration of large time series of volumetric images, such as those produced by the latest 3D + time fluorescence microscopy. They include integrated algorithms that allow researchers to interactively explore complex spatiotemporal objects at the scale of single cells or multicellular systems, almost in a real time manner. In practice, however, immersion of the user within 3D + time microscopy data represents both a paradigm shift in human-image interaction and an acculturation challenge, for the concerned community. To promote a broader adoption of these approaches by biologists, further dialogue is needed between the bioimaging community and the VR&AR developers.

13.
Opt Express ; 19(14): 12961-8, 2011 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-21747447

RESUMO

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. The method is found to work with both smooth and even completely random distortions. The experimental results are verified with MIIPS measurements.


Assuntos
Compressão de Dados/métodos , Interferometria/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Telecomunicações/instrumentação , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento
14.
Opt Express ; 19(10): 9000-7, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21643153

RESUMO

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. The electro-optical response from a nanocrystal provides local subwavelength sensor of quasi-static electric fields with potential applications in physics and biology. It also leads to a new sub-wavelength microscopy towards the nanoscale investigation of interesting phenomena such as nanoferroelectricity.

15.
Front Bioinform ; 1: 775379, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-36303735

RESUMO

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. Analyzing this type of experimental data is a complex task and presents unique challenges, where providing extra physical dimensions for visualization and analysis could be beneficial. Furthermore, whether highly noisy data comes from single-molecule recordings or segmented medical data, the necessity to guide analysis with user intervention creates both an ergonomic challenge to facilitate this interaction and a computational challenge to provide fluid interactions as information is being processed. Several applications, including our software DIVA for image stack and our platform Genuage for point clouds, have leveraged Virtual Reality (VR) to visualize and interact with data in 3D. While the visualization aspects can be made compatible with different types of data, quantifications, on the other hand, are far from being standard. In addition, complex analysis can require significant computational resources, making the real-time VR experience uncomfortable. Moreover, visualization software is mainly designed to represent a set of data points but lacks flexibility in manipulating and analyzing the data. This paper introduces new libraries to enhance the interaction and human-in-the-loop analysis of point cloud data in virtual reality and integrate them into the open-source platform Genuage. We first detail a new toolbox of communication tools that enhance user experience and improve flexibility. Then, we introduce a mapping toolbox allowing the representation of physical properties in space overlaid on a 3D mesh while maintaining a point cloud dedicated shader. We introduce later a new and programmable video capture tool in VR and desktop modes for intuitive data dissemination. Finally, we highlight the protocols that allow simultaneous analysis and fluid manipulation of data with a high refresh rate. We illustrate this principle by performing real-time inference of random walk properties of recorded trajectories with a pre-trained Graph Neural Network running in Python.

16.
Front Bioinform ; 1: 777101, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-36303792

RESUMO

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. While optimizing the visualization and interaction process in VR remains an active topic, one of the most pressing issue is how to utilize VR for annotation and analysis of data. Annotating data is often a required step for training machine learning algorithms. For example, enhancing the ability to annotate complex three-dimensional data in biological research as newly acquired data may come in limited quantities. Similarly, medical data annotation is often time-consuming and requires expert knowledge to identify structures of interest correctly. Moreover, simultaneous data analysis and visualization in VR is computationally demanding. Here, we introduce a new procedure to visualize, interact, annotate and analyze data by combining VR with cloud computing. VR is leveraged to provide natural interactions with volumetric representations of experimental imaging data. In parallel, cloud computing performs costly computations to accelerate the data annotation with minimal input required from the user. We demonstrate multiple proof-of-concept applications of our approach on volumetric fluorescent microscopy images of mouse neurons and tumor or organ annotations in medical images.

17.
Nat Commun ; 11(1): 5612, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154377

RESUMO

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. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns.


Assuntos
Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Cromossomos de Mamíferos/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Cricetinae , Drosophila , Camundongos , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Relação Estrutura-Atividade , Coesinas
18.
J Mol Biol ; 432(16): 4745-4749, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32512003

RESUMO

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. In DIVA's immersive virtual environment, users can view, manipulate and perform volumetric measurements on their microscopy images as they would to real physical objects. In contrast to similar solutions, our software provides high-quality volume rendering with native TIFF file compatibility. We benchmark the software with diverse image types including those generated by confocal, light-sheet and electron microscopy. DIVA is available at https://diva.pasteur.fr and will be regularly updated.


Assuntos
Imageamento Tridimensional/instrumentação , Realidade Virtual , Humanos , Microscopia , Software , Interface Usuário-Computador
19.
Biophys J ; 97(11): 2913-21, 2009 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-19948120

RESUMO

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. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed.


Assuntos
Corantes/metabolismo , Bicamadas Lipídicas/metabolismo , Membranas Artificiais , Imagem Molecular , Membrana Celular/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Potenciais da Membrana , Técnicas Analíticas Microfluídicas , Microscopia , Fenômenos Ópticos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Potenciometria , Compostos de Piridínio/metabolismo
20.
Nat Commun ; 9(1): 3181, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093638

RESUMO

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 H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis.


Assuntos
Proteínas de Ciclo Celular/química , Cromatina/química , Replicação do DNA , Histonas/química , Linhagem da Célula , DNA/química , Epigênese Genética , Genoma Humano , Células HeLa , Humanos , Hidroxiureia/química , Microscopia , Microscopia de Fluorescência , Chaperonas Moleculares , Nucleossomos/química , Fase S
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