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  Cid


  Director : S.L.SHORTE (sshorte@pasteur.fr)


  abstract

 

Established in 2001, the CID is a well equipped "technical-platform" facility providing expertise to aid researchers by designing, performing and analysing experiments using digital imaging microscopy. In particular, our focus is on dynamic imaging in living cells and tissues in order to gain insight on questions concerning cell biology, cell signalling, cell/tissue organisation and infectious processes. The facility is open to researchers inside and outside Pasteur, who benefit both from the wide variety of cutting-edge techniques offered, and our commitment to pursuing challenging projects requiring novel solutions through collaborative R&D efforts.



  report

cale

The Centre d'Imagerie Dynamique (CID) is a new technological platform at the Institut Pasteur comprising a part of the Plateaux Techniques initiative instigated by the Direction des Equipments et Technologies Stratégiques (DETS). Specifically, the purpose of the CID is to provide and develop high-level cellular imaging technology and expertise for addressing biological questions, which require visualisation and measurement of dynamic processes inside living cells. In addition, our role is to facilitate both national and international collaboration initiatives through, for example, scientific networks like the "European Light Microscopy Initiative" (ELMI). We are also committed to support specialised academic teaching courses hosted within Pasteur.

Albeit primarily a technical platform open to all, the CID is more specifically integrated into the Pasteur scientific community through a direct affiliation with the department of Cell Biology & Infection (P.Sansonetti), and has strong links within the departments of Developmental Biology (M.Buckingham), and Molecular Medicine (J.L.Virelizier). The research experience of our team covers extensively these domains (among others), and allows for us to provide both knowledgeable technical advice necessary for planning and execution of experiments, and insightful academic consultancy for establishing new approaches to specific biological questions. This combination of technical and academic research-engineering expertise makes the CID a highly specialized technical resource and as such affords a unique scientific service.

Note: the facility is (summer 2002) situated in an ergonomic, purpose built environment (Bât.Monod).

Examples of current projects/collaborations underway include a variety of approaches for 3-/4-D spatial/time analysis of subcellular protein distribution and dynamics e.g. Actin cytoskeleton changes induced by toxins in living cells (M.R. Popoff); Analysis of subcellularly localized actin-binding protein distribution and induction of actin polymerisation in living cells (B.Boeda, C.Petit); Effects of viral-protein over-expression on nuclear membrane dynamics & organisation, study of dynamics nuclear membrane invaginations (S. Münter, U.Nehrbass); Three-dimensional spatial analysis of FISH signals localized to chromosomes of parasite nuclei (L.H.Freitas-Junior, A.Scherf), Analysis of "immuno-synapse" structure and membrane dynamics in living cells (V.Das, A.Alcover), Effects of virus infection on synaptic vesicle recycling activity in primary neurons using fluorescent membrane markers (D.Gonzales, M.Brahic).

Equipment and Technical Support. The CID currently maintains twelve "state-of-the-art" microscopes, each customised for specific applications.

IMAGE ACQUISITION

(All systems are based around Zeiss motorized microscopes and PC software driven control)

Single Photon Confocal Microscopes:

Zeiss LSM510 — Ar/He/Ne laser, equipped for FCS, FRAP, FRET and 3-/4-D imaging

Zeiss LSM510 — Ar/He/Ne laser, equipped for 3-D imaging

Leica TCS-4D Kr/Ar laser (permenantly situated in Lwoff building)

Perkin-Elmer — Nipkow spinning disk system for fast confocal imaging in live cells.

2-Photon Confocal Microscope.

Zeiss LSM510 For "deep" in vivo and in situ 4-D tissue visualisation.

Conventional Light Excitation Digital Fluorescence Imaging Stations.

Three TILL "Fast" Imaging Systems equipped for quasi-real-time multi-dimensional imaging

Four Zeiss Multi-dimensional fluorescence time-lapse imaging systems.

IMAGE ANALYSIS

IRIX based Hardware/Software:

Two SGI Octane2 Workstations plus 1 SGI ORIGEN 3400 Server (IRIX 6.5)

Huygens/BitPlane Image processing software for 3-4-D reconstruction/deconvolution.

Microinformatics- 5 "high-end" PC's running various image analysis/processing and visualisation software for customised image processing/enhancement, morphological, numerical, algorithmic, and counting operations.

LIVE CELL/TISSUE PREPARATION, MANIPULATION & MAINTENANCE.

  • P2 cell-culture room
  • Tissue preparation room
  • 3 Bioptechs Live Cell Incubation Chambers
  • Evotek microscope based single cell sorting, and 3-D manipulation.

Activity turnover.

Currently, we host around 75 experimental sessions (each lasting 3-6hrs) per month and during 2002 expect this figure to rise to a peak of between 150-250/month.

Techniques for future development.

  • Single cell microinjection,
  • Infra-red laser micro-dissection;
  • Pulsed-"flash"-laser photolysis,
  • Four-dimensional deconvolution/reconstruction processing and object-tracking analysis,
  • Bioluminescence imaging and photon counting

Projects for future development.

  • Single cell activation of heat-shock promoter driven gene expression (in living, deep tissue sections and on cells in culture),
  • Quantitative "real-time" bioluminescence measurement of gene expression in single living cells using promoter driven luciferase, or colentrazine,
  • Measurement of molecular force generation at cell-substrate adhesion surface,


  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
       

PERRET, Emmanuelle, (Technican, eperret@pasteur.fr)

ROUX, Pascal, (Engineer, proux@pasteur.fr)

SHORTE, Spencer (Director, sshorte@pasteur.fr)


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