Core Facilities

Microscopy

Contact

Tristan Piolot
Tel : 01 44 27 15 96

Julien Dumont
Tel : 01 44 27 10 28

Image processing and analysis ; data storage:
Philippe Mailly
Tel : 01 44 27 14 21

Former member: Jérémie Teillon

The purpose of the CIRB microscopy core facility is to allow the most efficient access to standard and advanced imaging techniques for users of all levels of experience. It offers a wide range of conventional, confocal and 2-photon microscopes as well as image analysis resources and storage. Three dedicated engineers provide training and full technical support for the use of the instruments and for data analysis. The facility is open to all members of the CIRB and to external users.

Microscopy equipments

Spinning-disk confocal (CSU-X1) / FRAP / TIRF

Spinning-disk confocal

Purpose and capabilities

  • Confocal observation of live or fixed sample
  • TIRF microscopy and FRAP ability using the ROPER system
  • Environment chamber (temperature and C02 control) for live cell imaging
  • Capable of fast imaging (30 frame per second)
  • Very high sensitivity camera (Evolve EMCCD) and high resolution camera (Coolsnap HQ2)
  • Motorized XY stage for multi-positions acquisition

Specifications

  • Nikon Eclipse Ti inverted microscope with motorized XYZ stage
  • CSUX1-A1 (Yokogawa) spinning-disk scan head
  • 2 cameras for spinning-disk confocal microscopy:

- «Coolsnap HQ2», for high resolution imaging
- «Evolve», for fast and high sensitivity imaging

  • 1 camera for TIRF microscopy:

- «Evolve», for fast and high sensitivity imaging

  • Point, ROI and « on-the-fly » FRAP system
  • Lasers:

- 405 nm, 100 mW
- 491 nm, 150 mW
- 561 nm, 50 mW
- 642 nm, 25 mW

  • 4x, 10x, 20x, 60x, and 100x objectives and a 100x TIRF objective (NA=1.46)
  • Powered by Metamorph Premier 7.7

Spinning-disk confocal with wide field of view (CSU-W1)

spinning w1-Web

Purpose and capabilities

  • Confocal observation of live or fixed sample
  • Very large field of view thanks to the combination of the Flash 4 CMOS camera sensor and the Yokogawa CSU-W1 scanner unit
  • Environment chamber (temperature and C02 control) for live cell imaging
  • The Flash 4 v2 CMOS combines good resolution and a good sensitivity (82% QE at peak)
  • Motorized XY stage for multi-positions acquisition

Specifications

  • Zeiss Observer Z1 inverted microscope with motorized XYZ stage
  • CSU-W1 (Yokogawa) spinning-disk scan head
  • CMOS (Flash 4) camera with a 2048x2048 sensor
  • Lasers:

- 405 nm
- 445 nm
- 491 nm
- 561 nm
- 642 nm

  • 5x, 10x, 20x, 60x, and 100x objectives
  • Powered by Metamorph Premier 7.8

Leica SP5 Inverted confocal

Confocal Leica SP5

Purpose and capabilities

  • Confocal observation of fixed tissue by fluorescence
  • Acquisition of 3D z-stack, time-lapse, spectral scan
  • Motorized XY stage for multi-positions and mosaic acquisition (high definition images of large surfaces)

Specifications

  • Leica DMI6000 inverted microscope with motorized XY stage
  • Lasers:

- UV (405 nm), 50mW
- Argon (458, 477, 488, 496, 514nm), 200mW
- DPSS laser (561nm), 25 mW
- HeNe (633nm), 15 mW

10x, 20x, 40x, 63x and 100x objectives.
Powered by Leica Acquisition Software 2.3

Leica SP5 Upright 2-photon & confocal

Microscopy-Fig3

Purpose and capabilities

  • In vivo and fixed tissue observation by fluorescence
  • 1-photon and 2-photon excitation
  • Leica CFS microscope stand for stability and space to work on live small animals
  • High NA, long working distance, water immersion 25x objective for 2-photon live imaging (mouse, zebrafish, tissue explant)
  • Acquisition of 3D z-stack, time-lapse, spectral scan

Specifications

  • Upright Leica CFS microscope with 2 interchangeable stages:

- Motorized XY stage for mosaic acquisitions
- Motorized XY stage with high working distance for small animal observation

  • Lasers :

- Argon (458, 477, 488, 496, 514nm), 200mW
- DPSS laser (543nm), 1.5 mW
- HeNe (633nm), 15 mW
- Pulsed Ti :Sa laser for 2-photons imaging, Maitai (Spectra-Physics) tunable from 690 to 1040 nm, with pre-compensation system

  • Fast scanner, up to 30 frames/second in 512x512
  • AOBS system replacing classical dichroïc mirrors
  • Detectors:

-  Confocal:

- 2 classic PMTs
- 2 very sensitive hybrid detectors

-  2 photon:

- 2 hybrid NDD for reflected light
- 2 classic NDD for transmitted light

  • Objectives: 10x, 20x, 40x, 63x for confocal Imaging, and 25x water immersion for 2-photon imaging.
  • Powered by Leica Control Software 2.4

2 photon Scientifica

Purpose and capabilities

  • In vivo tissue and organism observation by fluorescence
  • 2-photon excitation
  • Scientifica microscope stand and stage allowing great stability and space to work on small animals
  • High NA, long working distance, water immersion 20x objective for 2-photon live imaging (mouse, zebrafish, tissue explant)
  • Acquisition of 3D z-stack, time-lapse

Specifications

  • Upright Scientifica microscope stand with 2 interchangeable object holders:

- One for small animals
- One for brain slices

  • Laser:

- Pulsed Ti :Sa, Maitai (Spectra-Physics) tunable from 690 to 1040 nm, with pre-compensation system

  • Resonnant scanner: 30 frames/sec at 512x512
  • Detectors:

- 1 classic PMTs
- 1 more sensitive GaAsP

  • Objectives: 16x, 20x water immersion for 2-photon imaging.
  • Powered by SciScan (Labview based solution)

Axiozoom/Apotome stereomicroscope

axiozoom-Web

Purpose and capabilities

  • Widefield or confocal-like observation of a wide range of sample sizes
  • Optical slices made possible with the Apotome structured illumination module
  • Very large field of view with the Flash 4 CMOS camera sensor
  • Continuous zoom ability (up to 16x) with a single objective
  • Motorized XY stage for multi-positions acquisition

Specifications

  • Zeiss Axiozoom.V16zoom microscope for large fields
  • Zeiss Apotome.2 module
  • CMOS (Hamamatsu Flash 4) camera with a 2048x2048 sensor
  • LED light source (X-CITE 110 LED)
  • Fluorescent filter sets for UV, green, red and fare-red stainings.

Video-microscope

Microscopy-Fig4

Purpose and capabilities

  • Wide field microscope for transmitted light, phase contrast and fluorescence (UV, GFP and TRITC) imaging
  • Fixed or live samples (cell culture and explants)
  • Environment chamber for temperature and C02 control allowing hypoxia experiments
  • Acquisitions of time-lapse, mosaics and multi-position

Specifications

  • Leica DMIRBE inverted microscope,with XY motorized
  • 4x, 10x, 20x, 63x objectives

- Coolsnap HQ (Roper Scientific) camera
- Controlled environment chamber: temperature, humidity, O2 and CO2
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Powered by Metamorph 6.6 software

Image analysis and data storage

5 image analysis workstations are available. 3 are running 64-bits operating system with 8 Go of RAM, allowing treatment and handling of large files exceeding 4 Go.

The following software packages are proposed:

- IMARIS, for 3D data visualization and quantification
- Stereoinvestigator for stereological analysis
- Metamorph Offline Premier 7.7, for segmentation of object and quantifications
- Huygens for widefield and confocal 3D stacks deconvolution
- Neurolucida for neuronal reconstruction and analysis
- ImageJ, Fiji and Nis Elements for data treatment and analysis
- IMOD for 3D reconstructions

Users will receive full training and assistance for these software packages.

2 mac Os X servers (10 and 4 To) and one OMERO server (20 To) software for visualization, management and analysis of biological microscope images.