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Fluorescence microscopy is an indispensable tool in biomedical research. New imaging techniques, in combination with powerful analysis software, have allowed scientists to move beyond the limits of optical resolution.

The Cellular Imaging Core Facility of the “Structure Fédérative de Recherche Necker Enfants Malades” is specialized in the visualization and analysis of the structure and dynamic processes at the cellular and tissue level until the organism level. Its mission is to provide biologists and practicians (internal or external to the Institute) advanced optical instruments and analytic tools accompanied with an expertise.

 

News on the platform :

Thanks to Imagine Institut financing, the Cell Imaging platform has just acquired a new confocal microscope equipped with STED technology ( Stimulated Emission Depletion) : a Leica TCS STED SP8 -3X with STED 660nm Laser confocal microscope .

The STED is a super- resolution method based on fluorescence confocal  imaging , in which images are acquired by scanning a focused light spot over a region of interest and  collecting  the fluorescence sequentially pixel by pixel. The main strengths of this technology are :
1 . Lateral resolution without any additional post- processing below 50 nm due to the vortex " donut "
2 . Intrinsic confocal optical sectioning , allowing the acquisition of three-dimensional structures
3 . Fast image acquisition of several images per second
4 . Live- imaging capabilities by using either fluorescent proteins or other fluorescent tags.

  • Nicolas Goudin  01 72 60 63 39 
    -  
  • Under Construction- Under Construction- Under Construction.

     

    Standard optical microscopy

    Wide field

    The widefield microscopy allows the acquisition of images and movies by a CCD camera on living or fixed samples. These samples can be cells, bacteria, yeast or tissue sections… The acquisitions can be made in three spatial dimensions (X, Y and Z), over time in transmitted or reflected light mode.

    Confocal Laser Scanning

    A confocal microscope is an optical microscope which has the property of producing images of very low depth of field (approximately 400 nm) called "optical sections". By positioning the focal plane of the lens at different levels of depth in the sample, it is possible to make a series of images from which a three-dimensional representation of the object can be obtained. The object is not directly observed by the user, he sees a reconstituted picture by computer.

    Advanced photonics microscopy

    TIRF (Total Internal Reflexion Microscopy)

    Total internal reflection fluorescence microscope or evanescent wave microscope is a special type of optical fluorescence microscope to examine a very thin slice of a sample (less than 200 nm thick), with a particular illumination mode: total internal reflection.The principle is to excite fluorescence on a very low depth, immediately adjacent to the glass interface (sample holder) / water (surroundings sample). The excitation is due to an evanescent wave. It is generated only when the incident light is totally reflected at the glass / water interface, which occurs only for a certain angle of incidence: the critical angle.

    With TIRFM we can see very selectively contact regions of the cell with its support and study with a very high resolution the morphology or the events occurring at the plasma membrane of living or fixed cells.

    Multiphoton microscopy

    The two-photon excitation consists in the absorption of two-photons simultaneously with a wavelength close to twice the one-photon excitation optimum. We use a pulsed laser in near-infrared frequencies. In this case, only the focusing point is excited by the laser beam. This system is considered as an important technological development for three main reasons:

    (i) There is no confocal iris (pinhole) in a two-photon microscope.

    (ii) The use of excitation light at a long wavelength (> 900 nm) provides greater penetration into the sample (up to 500 microns instead of 150 microns) offering the opportunity to work on thicker samples.

    (iii) The excitation of the fluorophores is limited at the focal point of the laser beam, the risk of photobleaching is reduced.

    In practice, the fluorescence emission performance is worse than a single photon confocal and the signal / noise ratio is lower. Thus, it shows little advantage for observing cultured cells or tissue sections (50-70 microns thick).

    FLIM (Fluorescence Lifetime Imaging Microscopy)

     

    Processing and image analysis

    The available software on both Necker and Broussais are:

    ImageJ and Fiji: Free software for processing and image analysis 3-4-5 D with many plugins and enabling the creation of customized scripts (macros).

    Imaris (Bitplane):  software for 3-4-5 D reconstruction and representation, quantification and image manipulation. It is interfaced with Matlab and Fiji.

    Metamorph: image manipulation software for opening images with the same interface than the Metamorph acquisition software. It’s also possible to analyze the images

    Zen (Zeiss): image manipulation software for opening images with the same interface than the acquisition software of Zeiss microscopes. It’s also possible to analyze the images.

    LAS AF (Leica):  image manipulation software for opening images with the same interface than the acquisition software of Leica microscopes. It’s also possible to analyze the images.

    NIS Elements: image manipulation software for opening images with the same interface than the acquisition software of Nikon microscopes. It’s also possible to analyze the images.

    Due to the relocation of a number of research teams on the Broussais hospital, the cellular imaging facility consists of two schedules:

    • One located at 96 Rue Didot, Pavillon Leriche, Gate 9, 75014 Paris
    • One at the Necker Enfants Malades hospital, Building Jean Hamburger Gate H1, 5th floor Door 537, 149 rue de Sèvres, 75015 Paris

    To contact us:

    Plateforme d'Imagerie Cellulaire de la SFR Necker UMS 24
    Hôpital Necker Enfants Malades
    Bâtiment Imagine
    RDC bas porte S9
    24 boulevard du Montparnasse 75015 Paris

    Tél : 01 42 75 42 67

    The missions of the platform are:

    • Assist users in the design and the implementation of their experience (protein labeling, choice of dyes, definition of controls …)
    • Provid users with advice and orientation towards microscopes adapted to their issues
    • Help users to carry out their experiments and gradually bring them to autonomy
    • Help users for processing and analyzing their data
    • Assist users in the interpretation and presentation of their results

    The staff of the platform supports

    • Development of imaging methods
    • Technical maintenance and metrology of the systems to ensure their daily operations.
    • Technology watch to follow the developments in light microscopy

    Contact is made exclusively by mail at

    Following this, we will set a date for an appointment to discuss the project. On this occasion, we will direct you to the most suitable equipment for your project.

     

    FEES:

     

    Long overnight experiments, Saturdays and Sundays , only 50% of the hours worked are invoiced.

     

    Metrology:

    Metrology is to achieve a number of measures in absolute values with standard steady readings in a predefined protocol to assess the functioning of a system over time and to ensure its proper functioning. Metrology is performed on each system platform at least twice a year.

    Applications for funding to develop the facility make it essential that the activity of the platform is explicitly recognized. This recognition may result in the articles by reference « image acquisition and image analysis were performed on the Necker Institute Imaging Facility » in the section Materials and Methods. And « the authors greatly acknowledge ___________of the Necker Institute Imaging Facility » in the acknowledgments.

    When the participation of a member of the platform has been decisive in the running of the project, the users include him in the list of signatories of the article.

    On the other hand, when using equipment funded by a foundation, association ... it should also be thanked (Imagine Foundation, ARC, FRM…).

    List of publications

    Abed J et al. Abnormal apical-to-basal transport of dietary ovalbumin by secretory IgA stimulates a mucosal Th1 response. Mucosal Immunol. 2013 Jul.

    Philippe et al. Dlg1 scaffolding protein participates with AP-1 and clathrin in forming Weibel-Palade bodies of endothelial cells. J Biol Chem. 2013 May.

    Khen-Dunlop N et al. Prenatal intestinal obstruction affects the myenteric plexus and causes functional bowel impairment in fetal rat experimental model of intestinal atresia. PLoS One, 2013 May.

    Lebreton C et al. Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides. Gastroenterology, 2012 Sept.

    Kurowska M et al. Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex. Blood. 2012 Apr.

    Putoux A et al.KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes. Nat Genet. 2011 Jun.

    Auffray C. et al. Patrolling blood monocytes that monitor blood vessels and tissues for damage and infection. Science 2007 Aug.

    Patey-Mariaud de Serre N. et al. Collagen alpha5 and alpha2(IV) chain coexpression: Analysis of skin biopsies of Alport patients. Kidney Int. 2007 August.

    Menager MM et al. Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat Immunol. 2007 Mar.