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LUMIC-Imaging and flow cytometry
Responsable : Dr. Didier Chatenay • Responsable Tech. : Dr. Susanne Bolte is the technical coordinator for this LUMIC candidature. 30%
Adresse : 4, place Jussieu, case postale 25
75005 Paris



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Presentation de la plate-forme

LUMIC is the UPMC network of 11 cellular imaging and flow cytometry core facilities. The facilities are located at the following research centers, Centre de Recherche des Cordeliers (CRC), Institute of Biology Paris-Seine (IBPS), Institut de Cardio-metabolisme et Nutrition (ICAN), Institut du Cerveau et de la Moëlle épinière (ICM), Institut de la Vision (IDV), Institut du Fer à Moulin (IFM), l'Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie (IMPMC), Laboratoire Jean-Perrin (LPJ) and at the medical school Pierre et Marie Curie (Pitié Salpêtrière, St. Antoine and Tenon hospitals). The key attributes of LUMIC is to bring together biologists, doctors and physicists by integrating facilities of the faculties of biology, medecine and t physics and providing technical support for fundamental and translational research in a large number of domains of biology and medical sciences.
Our facilities are equipped to analyze samples from the molecular to the whole organism level and with Angstrom to millimeter resolution. We provide an integrated offer of equipment and know-how to investigate minerals, lipids, proteins and their interaction partners, microorganisms and their association with minerals, cells, tissues, organs and whole embryos of mouse, chicken, Drosophila, zebrafish, ascidies, C. elegans, Xenopus and plants.

The methods employed are photonic and electron micoscopy, flow cytometry and cell sorting, mass-cytometry, multiplex analysis and molecular interaction studies. Our service offers include sample preparation, data acquisition, data processing and analysis in 4 core areas:

-Fluorescence imaging from the molecular level to the whole organism, imaging of live and fixed cells, tissues and small organisms.
-Live cell imaging, analysis of molecular dynamics in live cells (FRAP, FLIP, photoactivation and photoconversion FDAP), calcium imaging, biosensors
-Confocal super-resolution
-Macroscopic imaging
-Photomanipulation (photolesion and optogenetics)
-Spectral imaging (confocal microspectroscopy)
-Raman confocal imaging
-TIRF/PALM and STORM and single particle tracking
-molecularinteractions, FRET, FLIM
-Development of data analysis workflows

-Clarification of samples for confocal, two-photon and lightsheet microscopy
-Confocal and two-photon imaging of tissues, organs and embryos
-Light sheet microscopy
-Data visualization, processing and analysis with IMARIS and ARIVIS, Deconvolution (confocal, two-photon, spinning disc, wide field), Segmentation, development of spatial analysis in 3 dimensions (co-localization, tracking, ramification), 3D-reconstructions, virtual slides
-3D tomography in electron microscopy: electron tomography and cryo-electron tomography in TEM, serial milling in FIB/SEM and array-tomography in SEM

Transmission electron microscopy (TEM):
-Conventional sample preparation and observation in TEM: ultrastructure, immunocytochemistry and negative contrast
-High pressure or plunge freezing, freeze-substitution
-Cryo-microscopy in TEM
-Correlative light electron microscopy
-Chemical mapping and spectroscopy in TEM or in STEM-in-SEM with High Angle Annular Dark Field (STEM-HAADF),
-Energy-dispersive X-ray spectroscopy (XEDS),
-Electron Energy Loss Spectrometry or imaging (EELS/EFTEM)

Scanning electron microscopy (SEM):
-Conventional sample preparation and observation in high resolution SEM and cryo-SEM
-Conventional imaging in secondary or backscattered electrons
-Energy-dispersive X-ray spectroscopy and mapping (XEDS)
-Sample preparation: critical point drying, ion beam milling, freeze-fracturing and freeze-etching, metal coating.

Focus Ion Beam-SEM:
Sample preparation for TEM and Scanning-Transmission X-Ray microscopy (STXM), serial milling

- Multi-parametric flow cytometry (15-18 markers), data acquisition
- Tools for multi-parametric flow cytometry data analysis (Softwares and « know how »)
- Cell death, Cell cycle and Calcium Flux analysis by Flow Cytometry
- Cell sorting (P2 security level available on 2 sites, enrichment by automated magnetic cell sorting and FACS sorting, microparticles, rare events, cloning devices for up to six populations simultaneously).
-Analysis in mass cytometry (Cytoff, up to 30 markers), data treatment and cluster analysis (SPADE)
- High-multiplexing platform (detection/quantification of secreted proteins or protein from cell/tissue lysate).
- Development of Multiplex detection kits.
- Spectral imaging of multiplexed biomarkers in brightfield or fluorescence.
- Virtual multiplexing: image registration of immuno-labelled serial sections, including registration of virtual slides.
- High throughput brightfield or fluorescence imaging of tissue slides (slide scanner).
- Image analysis, including analysis of virtual slides and Tissue Micro Arrays
- Real Time Cell Analyzer (xCELLigence) ?
-xCELLigence Real Time Cell Analyzer MP and DP technology (real time analysis of cell adhesion, proliferation, invasion and migration by impedance-metry)

Moyens et equipements

Because the LUMIC network brings together biologists and physicists, we offer a particularly broad range of high-end microscopes and expertise. A number of complementary up-to-date photonic devices (12 confocals, 4 multiphoton microscopes, 9 spinning disc microscopes, a TIRF-microscope and a PALM/STORM microscope, 4 video-microscopes, 3 macroscopes, 4 laser-microdissection devices, 6 high throughput microscopes, 3 of them coupled with microfluidics and 4 light sheet microscopes, with two of them being prototypes for live imaging) are available for cellular imaging of living and fixed samples.
Flow cytometry and cell sorting can be carried out on 7 multilaser flow cytometers, 5 cell sorters, with two of them in P2-confinement. A mass-cytometer is accessible. This apparatus is one of two equipments accessible in an open access facility in the region. 2 Luminex Multiplex analysis systems, a Real Time Cell Analyzer (xCELLigence MP and DP) as well as a Biacore system for molecular interactions are also available.
Concerning electron microscopy, we provide a unique instrument park. We have made the strategic decision to place a novel High resolution FESEM purchased by IBPS on the IMPMC-site in order to foster synergies between biologists and physicists. Our facilities are thus equipped with 2 Scanning Electron Microscopes (SEM) for imaging at room temperature or cryo and chemical analysis (EDS) and 1 FIB/SEM for serial imaging or sample preparation for TEM or STXM. Furthermore, it includes 4 Transmission Electron Microscopes (TEM) to observe standard sections of samples (80-120 kV with tungsten or LaB6), or to perform electron tomography or cryo-TEM and chemical analysis (EDS & EFTEM) at conventional and high resolution (200 kV with LaB6 or FEG). These instruments are associated to sample preparation equipment that covers a large spectrum of applications in EM applied to biological or material sciences (cryofixation, ultramicrotomy, freeze fracturing, polishing, critical point drying, metal coating), allowing to characterize raw materials and molecules and to investigate their applications in materials, environment and health.

Leica TCS SP5 AOBS, inverted, Hybrid detector, resonant scanner, 2005, upgraded 2011 (IBPS)
Leica TCS SP5 AOBS, upright, hybrid detector, 2007 upgraded 2011 (IBPS)
Olympus FV1000, upright, 2007 (ICM)
ZEISS, LSM710, inverted, 2009 (CRC)
Olympus FV1000, inverted, 2009 (IDV)
Olympus FV1000, upright, 2010 (IDV)
Leica TCS SP5 AOBS, upright, Hybrid detector, 2010 (IFM)
Olympus FVi10x, inverted, 2011 (IFM)
Leica SPE, upright, 2011 (IBPS)
Olympus FV1200, inverted, 2014 (IDV)
Leica TCS SP8 AOBS, inverted, hybrid detector, 2016 (ICM)
Olympus FV1200, upright, GaAsP detector, 2016 (ICM)

ZEISS 710 NLO, upright, GaAsP detector, 2009 (ICM)
Leica TCS SP8 MP5, upright, Hybrid detector, resonant scanner, 2011, upgraded 2016, (IBPS)
Leica TCS MP5, upright, Hybrid detectors, resonant scanner, 2011 (IFM)
3I, upright, resonant scanner, Holography, GaAsP-detectors, 2016, (ICM)

Upright Spinning disc microscope Roper/Leica, 2004, (IDV)
Inverted Spinning disc microscope Roper, Metamorph, 2008, (IBPS)
Inverted Spinning disc microscope/Leica, 2008, (IFM)
Inverted Spinning disc microscope Roper/Leica, 2008, (UMS-StA)
Upright Spinning disc microscope Roper/Leica, 2010, (IDV)
Upright spinnnig disc microscope with FRAP-photoactivation, 2013, (IBPS)
Inverted microscope Olympus IX83 + DSD2 (Andor) (Tenon), 2014, upgraded in 2017, (Tenon)
Inverted Spinning disk microscope Andor Leica Yokogawa X, 2016, (ICM)
Upright Spinning disk microscope Yokogawa W, 2017, (ICM)

LAVISION Biotech Ultramicroscope with far-red laser, 2011, (IDV)
LAVISION Biotech Ultramicroscope, 2017, (ICM)

Fluorescence/brightfield slides scanner,Hamamatsu, 2010, (IDV)
Fluorescence/brightfield slides scanner, Axioscan Z1, Zeiss, 2015, (CRC)

2 lightsheet microscope prototypes, 2015, (LJP)
Raman Confocal Microscope and Raman Tweezers, 2013, (LJP)
3 optical microscopes couples with Microfluidics devices, 2014-2016, (LJP)

Automate immunhistochimie Dako, 2010, (UMS-StA)
Tissue microarray, 2001, upgrade 2011, (UMS-StA)
Automated IHC/ISH research slide staining system Ventana Discovery XT, Roche, 2015, (CRC)

Automated Multispectral Imaging Microscope, Vectra, PerkinElmer, 2016, (CRC)
Multispectral Imaging Microscope, Mantra, PerkinElmer, 2015, (CRC)
High through-put cell imager, BD Cell Signaling Pathways 855, Becton Dickinson, 2011, (CRC)

Laser Microdissection microscope, AS-LMD, Leica, 2005, (CRC)
Laser Microdissection PALM Zeiss, 2005, (UMS-StA)
Laser microdissection Arcturus, 2015, (IBPS)
Laser microdissection Leica DM6500B , 2010 (IDV)

Transmission electron microscope (TEM) CM100 Philips equipped with a CCD GATAN Orius camera, 1996, (IFM)
Transmission electron microscope Jeol 1010, 100kV and MégaViewIII camera (Tenon), 1997 et 2007, (Tenon)
Transmission Electron Microscope JEOL JEM 2100F 200kV + Gatan GIF 2001 + Gatan US4000 CCD camera + STEM-HAADF+BF+ Jeol XEDS detector + cryo-TEM devices + tomography, 2003, (IMPMC)
Scanning Electron Microscope Zeiss Ultra 55 + Bruker Quantax XEDS system, 2006, (IMPMC)
Focused Ion Beam Zeiss Neon 40EsB + Bruker Quantax XEDS system + Atlas 3D Tomography Module, 2006, (IMPMC)
Transmission Electron Microscope JEOL JEM 2100 200kV + Gatan US1000 CCD camera + STEM&
8209;BF + Jeol XEDS detector + cryo-TEM devices + tomography (bright field), 2007, (IMPMC)
Transmission electron microscope JEOL JEM 2100 80 - 200kV + Gatan Gif Tridiem camera + tomography and cryo-TEM devices, 2007, (IBPS)
Transmission electron microscope Hitachi 120kV, 2010, (ICM)
High resolution FESEM (GeminiSEM 500, Zeiss) with STEM detector, cryo-stage (Leica microsystems- and Atlas5 Array tomography (Zeiss), 2015, (IBPS)

Freeze fracturing apparatus and coaters Baltec BAF400T and ACE600 with VCT100 (Leica Microsystems), 1994 and 2015, (IBPS)
HPM 100 High pressure freezing machine (Leica Microsystems), 2010, (IBPS)

Flow cytometer analyser, LSR II, Becton Dickinson, 2006, (CRC)
Flow cytometry analyzer LSRII (Becton Dickinson), 2007(UMS-StA)
Flow cytometer analyzer FC500 (Beckman Coulter), 2005 (IDV)
Analyseur-Sorter 5 lasers confinement L2, 2007, (UMS-PS)
Flow cytometry analyzer Gallios (Beckman Coulter), 2011, (UMS-StA)
Cell sorter MoFlo-Astrios standard (Beckman Coulter), 2011, (UMS-StA)
Analyseur 5 lasers, 2011, (UMS-PS)
Flow cytometer cell sorter, ARIA III, Becton Dickinson, 2012, (CRC)
Flow cytometer cell sorter, Influx, Becton Dickinson, 2013, (CRC)
Flow cytometer Miltenyi MACSquant Analyzer 10 ,2013,(ICAN)
Cell sorter Beckman Coulter MOFLO Astrios eQ , 2014, (ICAN)
Automatic Magnetic cell sorter, AutoMacs, Miltenyi, 2014, (CRC)

CYTOF II upgraded in Hélios, 2014, (UMS-PS)

High-multiplexing platform, FLEXMAP3D, Luminex, 2013, (CRC)
Magpix Multiplex system, 2013, (ICAN)
Magpix Multiplex system, 2015, (IDV)

Biacore 3000 (GE Healthcare) , 2008, (IBPS)


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