The IMRB imaging facility provides expert guidance and support to IMRB staff and to external users for histology and microscopy, from the sample preparation to the image acquisition.
The IMRB imaging facility have been certified ISO 9001 since April 2014. This certification guarantees a set of requirements concerning the quality management in the facility. This will facilitate industrial partnerships.
The facility provides all the equipment needed from the sample preparation for histology to observation in microscopy.
The IMRB imaging facility provides two services:
1) Sample preparation
2) Optic microscopy
1) Sample preparation department
This department includes equipment for:
a) Option microscopy preparation;
b) Electron microscopy preparation
a) Option microscopy preparation
The IMRB imaging facility provides either an all-inclusive service (from the sample preparation to the sectioning) or a do it yourself service (after a training provided by the IMRB imaging facility staff). Booking is achieved at obm.inserm.fr.
The tissue sample fixation must be done with formol. The sample size must not exceed 4mm in thickness by 2cm in width by 2,5cm in length.
The preparation begins with the dehydration and paraffin infiltration of the samples. These steps are performed by a Milestone Logos automaton to obtain the best impregnation. Then the samples are embedded in paraffin on a Thermo Scientific HistoStar station.
From left to right: impregnating PLC Logos, paraffin fountain HistoStar, microtome Shandon Finesse
3 to 30µm sections are obtained with a manual microtome Shandon Finesse 325. They can be can be recovered on Super-Frost+ microscope slides (for immuno-chemistry stainings), or on untreated microscope slides (for ulterior histological stainings). There is also a freezing plate Histostar to cool down the paraffin embedded samples and a heating plate to smooth the section. The IMRB imaging facility also provides a Leica CM3050S cryo-microtome allowing frozen sectioning at -40°C and from 5 to 100µm thichkness.
From left to right: Microtome Finesse and cold plate, cryo-microtome.
Sections can be stain with Mayer’s hemalun and eosin. Nucleus appears in purple and the cytoplasm in pink. The most common histological stainings are also available upon request.
b) Electron microscopy preparation
The facility achieve also all operations to obtain grids for Transmission Electron Microscope (TEM)
-Chemical fixation and EPON Resin embedding
As the chemichal fixation is performed with glutaraldehyde. The tissue sample size MUST BE less than 1mm x 1mm x 1mm. Prior to the resin embedding process, the samples are exposed to a second fixative: Osmium tetroxyde.
2µm-sections are then stained with a mixture of Azure II/methylene blue allowing the selection of a region of interest to complete ultra-thin sections.
-Grid production for TEM
3 or 4 sections from 60 to 150 nm thick are set on a copper grid. After staining with uranyle acetate/lead citrate, the grids are ready for observation.
Grid for TEM
EPON resin with different tissue appearing in black
Observations are performed on a transmission electron microscope JEOL 100CXII at the LISA microscopy facility. To do so, please contact Patrick Ausset (Patrick.Ausset@lisa.u-pec.fr). http://www.lisa.univ-paris12.fr/fr/instruments/33-analytical-instruments-laboratory/117-microscopes
2) Optical Microscopy Department
The optic microscopy department is dedicated to microscopy imaging of histological or fluorescent sample.
3 microscopes are available:
a) Axioplan 2 microscope
b) AxioImager M2 microscope
c) LSM 510 confocal microscope
The Axioplan 2 is dedicated to histology imaging. The setting includes a large scale of magnification thank to several objectives (Plan-NeoFluar x5/0,16, Acroplan x10/0,25, x20/0,45. Plan-Neofluar x40/0,75, x63/1,25 oil immersion) and a Zeiss Mrc camera allowing the image acquisitions through the Zeiss Zen 2012 software.
The AxioImager M2 is dedicated to fluorescence imaging. The setting includes 4 fluorescence cubes (DAPI, A488/FITC, A547/TRITC, To-Pro3/Cy5) for the most used fluorescent molecules in biology research, 4 different objectives (Acroplan x10/0,25, x20/0,45, Plan-Neofluar x40/0,75) and a monochrome Zeiss Mrm camera allowing the image acquisitions through the Zeiss Zen 2012 software.
The LSM 510 Meta is set up on an inverted microscope Zeiss Axio Observer Z1. The setting includes a motorized stage and a thermostated chamber, 6 different LASER lines (458nm, 477nm, 488nm, 514nm, 543 nm, 633nm) covering a large array of the fluorescent molecules used in biology research.
Fluorescence distribution (2D or 3D), z-stack acquisition, co-localisation, spectral analysis (fluorescence spectra unmixing) and applications on living cells (live-microscopy, FRAP) can be performed on this setup.
Since April 2014, the imaging platform is ISO 9001 which will facilitate its opening for industrial partners.
ISO 9001 defines a set of requirements concerning the implementation of a quality management system in an organization, regardless of size and industry.
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