DONATE MAINS

HIST1

The Imaging Core Facility of the Institut de la Vision provides access to a broad range of biological imaging solutions for life sciences research. Light microscopes and analysis expertise of the facility members are made available to both academia and industry on a user cost basis. Facility staff is available to teach, train, consult, advise and collaborate. The staff works closely with investigators to provide technical support by developing procedures and protocols to ensure state-of-the-art imaging.

Professional scientists are available to consult with research team members on the design and implementation of imaging experiments; to assist with data acquisition, analysis, and presentation; and to educate and train on the theory and practice of the technologies at hand.


Expertises

  • Light microscopy : widefield, confocal and live imaging ;
  • Image analyis : ImageJ, Imaris ;
  • Configuration, calibration and troubleshooting of microscopy and imaging instruments ;
  • Cellular biology: tissue and cell preparation, immunostaining.

Equipements

Upon agreement about rules and guidelines as well as a practical training, users have free access to various sophisticated optical microscopy imaging systems and analysis workstations.

Available imaging modalities are:

 

Confocal laser scanning microscopy:

Confocal microscopy enables acquisition of three-dimensionally resolved fluorescence images by collecting emissions only from the focus of a scanned laser beam.  Our state-of-the-art confocal microscopes have a large range of excitation laser lines, emission color, and environmental controls.
Confocal microscopy can be used to locate fluorescent objects with sub-micron resolution in live or fixed cells, engineered or cultured tissues. Samples can be fixed or live, sectioned or unsectioned, stained or unstained depending on the application.  After an individual training and consultation session, researchers are given access to schedule usage of the instruments.    Assistance with imaging and experimental design is available during normal business hours.

  • An Upright confocal microscope Olympus FV-1000 with the following wavelengths: 405 (diode), 440, 488 (Argon), 515 (Argon), 559 (diode) and 635 nm (diode). This set up is configured with a motorized stage for multi-location and tile-image recording. Two GaAsp detectors are available, allowing a more sensitive imaging for either Green + Red, Red +Far Red or Cyan + Red. Available objectives: 10X dry (NA 0.4), Super10X (water dipping, NA0.6), 20X oil (NA 0.85), 40X oil (NA 1.30), super corrected 60X oil (NA 1.40) and 100X oil (NA 1.30).
  • An Inverted confocal microscope Olympus FV-1000 with the following wavelengths: 405 (diode), 440, 488 (Argon), 515 (Argon), 559 (diode) and 635 nm (diode). This set up is configured with a motorized stage for multi-location and tile-image recording. This setup is ready for live imaging owing to a 37°C incubation chamber and a CO2 enriched environment. Available objectives: 4X dry (NA 0.30), 10X dry (NA 0.4), 20X dry(NA0.75), 20X oil (NA 0.85), 40X oil (NA 1.30),60X oil (NA 1.40).
  • An Inverted confocal microscope Olympus FV-1200 with the following wavelenghts: 405 (diode), 440, 488 (Argon), 515 (Argon), 559 (diode) and 635 nm (diode). This set up is configured with a motorized stage for multi-location and tile-image recording. Two GaAsp detectors are available and allow a more sensitive imaging for either: Green + Red, Red +Far Red or Cyan + Red. Available objectives: 4X dry (NA 0.30), 10X dry (NA 0.4), 20X oil (NA 0.85), 40X oil (NA 1.30), super corrected 60X oil (NA 1.40) and 40X silicon immersion (NA 1.25).

Spinning disk confocal microscopy:

  • Live cell time-lapse imaging set-up for 3D-imaging with a Yokogawa CSU-X1 spinning disk scan head, mounted on a DM6000 upright Leica microscope. Images are acquired using a CCD CoolSnap HQ2 (Roper) camera. This setup is ready for live imaging owing to a 37°C incubation chamber and a CO2 enriched environment. Available laser lines are: 440, 515, 491 and 561 nm. Available objectives: 10X,  20X and 40X water-dipping. Note that 20X and 40X dry long working distance objectives are also available (on demand). This setup is associated with an additional second one, providing the opportunity to image two samples simultaneously, i.e. wild type and mutant experiments at the same time.

Sheet plane illumination imaging:

  • A commercial light sheet microscope (Ultramicroscope, Lavison Biotec) is available. This system creates a thin (4 µm) light sheet to illuminate sample from the side while imaging the excited plane by a microscope perpendicular to the light sheet; moving the sample through the light sheet generates a 3D image stack. It can image samples ranging in size from 1 cm to a few µm. The Ultramicroscope is equipped with three different laser lines: 488, 561 and 635 nm and a CMOS camera. Note that this imaging technique requires a tissue clearing.

Virtual slide scanner:

  • A Hamamatsu slide scanner 2.0 HT is available and can process up to 210 slides. It can image both brightfield (e.g. H&E slides) and up to three to four color fluorescent samples (405, 488, 560 and 633 nm excitation or 440, 515 and 560 nm) to form montages of the whole sample, or multiple samples on each slide. The resulting montages are fully scalable (one can zoom in in a 'Google Maps'-like fashion) and can be exported as a whole. The recent purchase of dedicated analysis software (MorphoStrider) will greatly help users to quantify and export a variety of image file formats. It can be found on the 'off-line' workstation for image analysis functions to analyse/quantify the images. Note that this is not a free access instrument, its usage requires the presence of the responsible engineer.
  • Cell imaging set-up for ratio imaging Built around an upright Nikon Ti80 microscope the calcium imaging setup is equipped with a Hamamatsu ORCA-03G and a Lambda DG4 for rapid switching of the excitation wavelengths. This setup is designed primarily for time-lapse high-speed imaging of intracellular calcium in living cells using Fura-2 but commonly filter sets are also available for classical fluorescent dyes.  Available objectives: 10X, 20X and 40X water-dipping.
  • Wide field microscopes; Wide-field microscopes are classical light microscopes with light source and beam paths optimized for fluorescence microscopy. Our systems are also configured for transmitted light techniques including differential interference contrast (DIC) and phase contrast.

Three upright microscopes:

  • A Leica DM5000 stand, equipped with a color camera CollSNAP (Roper) and the following filter cubes : A4 (DAPI or Alexa 350), L5 (green fluorescence : eGFP or Alexa 488), TX2 (red fluorescence : RFP or Alexa 594) and GR (Green and Red).
  • A Leica DM6000 stand, equipped with a black and white  CoolSNAP HQ (Roper) and the following filter cubes : A4 (DAPI or Alexa 350), L5 (green fluorescence : eGFP or Alexa 488), Y3 (red fluorescence : RFP or Alexa 594), Y5  (far red like Alexa 647),  CFP et YFP. A Y7 filter is also available (on demand) to image infrared fluorochromes.
  • A Zeiss M1 stand, equipped with a color camera (Zeiss) and the following filter cubes : DAPI (DAPI or Alexa 350), Green (green fluorescence : eGFP or Alexa 488), Red (red fluorescence : RFP or Alexa 594), Far red  (far red like Alexa 647).

A more specialized instrument : an automated acquisition setup:

  • A morphometry setup built on an inverted Nikon TiE stand, with a CoolSNAP HQ2 camera (Roper), a CoolSNAP CF2-Color (Roper), an Optigrid module and a motorized stage for multi-location and tile-image recording using a Metamorph 7.7 scanslide module. This instrument is equipped with classical filter cubes (DAPI, FITC and Texas Red), fast imaging dedicated cubes (Live/Dead and DAPI/TxRed) and drived by Metomorph 7.7 associated with additional options (Screening, Scanslide, Neurite outgrowth and Count nuclei).

Available analysis modalities are:

  • Imaris ;
  • To image and quantify 3D samples ;
  • MorphoStrider ;
  • To analyze virtual slide scanner images ;
  • ImageJ / FIJI ;
  • To analyse all scientific images produced on the core facility and beyond.


TEAM

Alain

Alain Chedotal

stephane

Stéphane Fouquet


Alain Chedotal
Scientific supervisor

Email: alain.chedotal@inserm.fr


Stéphane Fouquet
Technical supervisor
Implementation of image analysis courses and tutorials
Consultation and training of new facility users.
Scientific and technological survey.
Core facility management related skills:

  • Executive project manager and establishment/optimization of protocols
  • Budget reporting and billing management
  • Development and management of the online reservation/booking system of the imaging facility instrumentation
  • Data back up management
  • Grant application writing and reporting
  • IDV technical representative in the Sorbonne University imaging network

Associated background:

  • Multi-color cytometry
  • Immunology
  • Cellular and molecular biology, biochemistry technics

Ph.D. in cellular biology and physiology

Email: stephane.fouquet@inserm.fr