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Cell and tissue imaging - UMR 3215 (pict-IBiSA)

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Leader: Olivier Renaud


videomicroscopy, confocal microscopy, cell ablation, developmental imaging, image processing and analysis

The imaging facility of the Unit of Developmental Biology and Genetics (UMR3215 CNRS / U934 INSERM) is a common service of optical microscopy and image analysis, located in the Developmental Biology and Cancer building on the Institut Curie's Paris campus. This service is part of the Platform for Cell and Tissue Imaging (PICT-IBiSA) of the Institute.


Live imaging is an asset for biological research
Fluorescence optical microscopy has become an essential imaging technique for research in biology, especially in the field of developmental biology. Recent technological progress have enabled the development of faster microscopes offering higher resolution to collect tridimensional images of living biological samples.In parallel, important progress have been made in the development of stable and bright fluorescent probes (see Fig. 1), leading to a breakthrough for in vivo tracking of individual cells.

These combined technologies enable noninvasive imaging of living cells, tissues and embryos, without disturbing their development. Imaging of living subjects provides better understanding of biological processes during development and illnesses, such as cancer.


 Fig.1: Image of 3.5 dpc mouse blastocyst obtained using spinning disk confocal microscopy. © Mounia Guenatri (D. Bourc'his team).Fig.1: Image of 3.5 dpc mouse blastocyst obtained using spinning disk confocal microscopy. © Mounia Guenatri (D. Bourc'his team).


The missions of the imaging facility are as follows:

  • to provide advanced technologies and expertise in optical microscopy and image analysis to the researchers of the Institut Curie unit and external researchers,
  • to provide users with training, help and advice,
  • to maintain a leading technological facility,
  • to perform technical developments and to design technologies and software,
  • to collaborate in scientific projects,
  • to teach and to pass over knowledge.


    Fig.2: Follicules ovariens de drosophileFig.2: Follicules ovariens de drosophile







The imaging service of the Unit of Developmental Biology and Genetics works in close collaboration with the unit's research teams, in order to provide them with tools dedicated to cell imaging of biological processes during the development of living organisms (see photo on Fig. 2).

Multiple difficulties have to be overcome to offer highly precise tracking of marked proteins over periods of time (for several hours or even several days), while maintaining spatial information (position of the protein in the cell, the tissue or the animal) and keeping the sample alive (see film).

Cell division

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Cell division in Drosophila epithelial tissue observed using confocal laser scanning microscopy over time. In green, one can track E-cadherin protein tagged with a Green Fluorescent Protein, which plays an important role in cellular adhesion


Advanced technology

 Recently developed fluorescent probes, in particular those developed by 2008 Chemistry Nobel laureates (Osamu Shimomura, Martin Chalfie et Roger Y. Tsien), are useful for long-term analysis, since they offer greater photostability over time and better luminosity. In addition, the use of microscope incubators enables to maintain organisms in physiological conditions through temperature control, CO2 percentage and humidity control. Finally, the development of more advanced microscopes enables in particular to collect the light emitted by these fluorescent probes, even when they are located deep in the sample, using powerful laser sources and highly-sensitive detectors.

Otherwise, resolution remains a limiting factor in optical microscopy, for precise observation of subcellular structures, such as nuclear proteins.The lateral resolution of a confocal microscope is approximately 250 nanometers (0.000000250 meters), which corresponds to the size of small bacteria (for ex. Mycoplasma), while the size of the structures we wish to observe is very often smaller (for ex. proteins, microtubules). A considerable breakthrough is now underway with the joint efforts of international physics, optics, chemistry and biology laboratories. The physical limitations in the field of optics are currently being overcome through the development of new technological approaches and intensive computational image processing. For example, structured-illumination microscopy enables to reveal interferences between the sample and the light source (Moiré effect). Another approach consists in using the physical characteristics of fluorescent probes to reduce the optical impulse response of the microscope in STED microscopy (“Stimulation Emission Depletion Microscopy”). Today these techniques are central to improving the resolution and thus the possibility of achieving a finer level of observation of living organisms.

Key publications

  • Year of publication : 2014

  • A new level of chromosome organization, topologically associating domains (TADs), was recently uncovered by chromosome conformation capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model's predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.
  • Year of publication : 2012

  • In eukaryotes transcriptional regulation often involves multiple long-range elements and is influenced by the genomic environment. A prime example of this concerns the mouse X-inactivation centre (Xic), which orchestrates the initiation of X-chromosome inactivation (XCI) by controlling the expression of the non-protein-coding Xist transcript. The extent of Xic sequences required for the proper regulation of Xist remains unknown. Here we use chromosome conformation capture carbon-copy (5C) and super-resolution microscopy to analyse the spatial organization of a 4.5-megabases (Mb) region including Xist. We discover a series of discrete 200-kilobase to 1 Mb topologically associating domains (TADs), present both before and after cell differentiation and on the active and inactive X. TADs align with, but do not rely on, several domain-wide features of the epigenome, such as H3K27me3 or H3K9me2 blocks and lamina-associated domains. TADs also align with coordinately regulated gene clusters. Disruption of a TAD boundary causes ectopic chromosomal contacts and long-range transcriptional misregulation. The Xist/Tsix sense/antisense unit illustrates how TADs enable the spatial segregation of oppositely regulated chromosomal neighbourhoods, with the respective promoters of Xist and Tsix lying in adjacent TADs, each containing their known positive regulators. We identify a novel distal regulatory region of Tsix within its TAD, which produces a long intervening RNA, Linx. In addition to uncovering a new principle of cis-regulatory architecture of mammalian chromosomes, our study sets the stage for the full genetic dissection of the X-inactivation centre.
  • Year of publication : 2011

  • Confocal live imaging is a key tool for studying cell behavior in the whole zebrafish embryo. Here we provide a detailed protocol that is adaptable for imaging any progenitor cell behavior in live zebrafish embryos. As an example, we imaged the emergence of the first hematopoietic stem cells from the aorta. We discuss the importance of selecting the appropriate zebrafish transgenic line as well as methods for immobilization of embryos to be imaged. In addition, we highlight the confocal microscopy acquisition parameters required for stem cell imaging and the software tools we used to analyze 4D movies. The whole protocol takes 2 h 15 min and allows confocal live imaging from a few hours to several days.
  • The conventional approach for microscopic 3D cellular imaging is based on axial through-stack image series which has some significant limitations such as anisotropic resolution and axial aberration. To overcome these drawbacks, we have recently introduced an alternative approach based on micro-rotation image series. Unfortunately, this new technique suffers from a huge burden of computation that makes its use quite difficult for current applications. To address these problems we propose a new imaging strategy called bi-protocol, which consists of coupling micro-rotation acquisition and conventional z-stack acquisition. We experimentally prove bi-protocol 3D reconstruction produces similar quality to that of pure micro-rotation, but offers the advantage of reduced computation burden because it uses the z-stack volume to accelerate the registration of the micro-rotation images.
  • Year of publication : 2010

  • Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises.
  • The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal "axial tomography," this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells.
  • Lu/BCAM, the unique erythroid receptor for laminin 511/521, interacts with the erythrocyte membrane skeleton through spectrin binding. It has been reported that Hereditary Spherocytosis red blood cells (HS RBC) exhibit increased adhesion to laminin. We investigated the role of Lu/BCAM-spectrin interaction in the RBC adhesion properties of 2 splenectomised HS patients characterized by 40% spectrin deficiency. Under physiological flow conditions, HS RBC exhibited an exaggerated adhesion to laminin that was completely abolished by soluble Lu/BCAM. Triton extraction experiments revealed that a greater fraction of Lu/BCAM was unlinked to the membrane skeleton of HS RBC, as compared to normal RBC. Disruption of the spectrin interaction site in Lu/BCAM expressed in the transfected K562 cell line resulted in a weakened interaction to the skeleton and an enhanced interaction to laminin. These results demonstrated that the adhesion of HS RBC to laminin was mediated by Lu/BCAM and that its interaction with the spectrin-based skeleton negatively regulated cell adhesion to laminin. Finally, the results of this study strongly suggest that the reinforced adhesiveness of spectrin-deficient HS RBC to laminin is partly brought about by an impaired interaction between Lu/BCAM and the membrane skeleton.