PICsl-FBI photonic- electronic Imaging Facility IBDM Imaging - PowerPoint PPT Presentation

PICsl-FBI photonic- electronic Imaging Facility

IBDM Imaging facility 200 researchers + 20 Quality process Service industrials Video-microscopy Tcn 11 video-microscopes scientific supervisor CNRS Pierre-François Lenne + DR2 CNRS 4 Master service Degree’s Confocal 4 confocal, Training Technical Metrology 3 spinning disk supervisor Operating Cédric Matthews project machining/ Budget IR2 CNRS computer-aided design / microscopy 2014 machining AI CNRS 100 000 euros Two photon microscopy Service / Project development 2 two photon Capital Budget Cédric Matthews Image processing since 1 two photon , 1macro- confocal 2009 1 million euros 1 IR , 1 AI , 1 TCN 22 systems

Main Equipments Two photons Spinnind disk Confocal Video microscopes 1 nikon high 1 Roper- 2 LSM 510 meta speed + script yokogawa X1 with 3 apotomes up to 1 LSM 510 2 cameras evolve date 1 zeiss 7 MP with 1 LSM 780 5 detectors 1 roper- 1 1 nikon yokogawa CSU 10 environnemental 1 zeiss 510 NLO macroconfocal dedicated to video-microscope inverted electrophysiology 3 microscopes

New Equipments High Confocal Déconvolution Technological resolution transfert from LSM 510 UV Treatment microscope Lenne team : meta upgrade Huygens PALM STED software Spinning disk microscope Specific dyed (team Lecuit) Omero antibodies database for Camera pixel images and size 8µm assistance

Available Techniques dynamic processing and imaging and Prototype systems images analysis contrast • ImageJ- IcY • Turning machine • Videomicroscopy • FRET • Automatisation • Milling • Confocal • FRAP of analysis • 3D printing • Two Photon • FCS • Matlab (coming soon) • Macro confocal • Absorption • Huygens • STED • Fluorescence deconvolution • SHG • Depletable dyes

Our Expertise Vocational training Associated to systems IBDM training • CNRS / INSERM • 5 different trainings : • Image processing • CNRS - company • Image analysis (ImageJ) • Metrology • Image analysis (ImageJ) • Macro – development • Quality control (dyes) • Imaging and quality • confocal Microscopy • Master's degree tutoring control • two photon Microscopy • Competitive Bidding publication • High resolution • computer-aided design / machining

Distribution of systems use videomicroscope confocal two photons STED 4280 hours of use from January to June 2014

Distribution of system trainings spinning LSM 780 LSM 510 video STED 11 individual trainings on video-microscopes and 15 on other microscopes from January to June

General overview

Trainings • We did : – ImageJ, ICY (Institut Pasteur) , PhD program • Coming : – Image J image treatment, deconvolution, - to schedule with CIML, IBDM, INMED – Microscopy theoretical aspect: video, confocal , high resolution (CIML, IBDM, INMED) – PhD program-labex inform (january) – training ICY (CIML,IBDM,INMED)

Recruitments • 2014, Master 2 intern from Nice Sophia University imaging master. • 2012-2014 , Engineer apprenticeship • July 2014-2016 : Remi Flores-Flores, 50 % imaging core facility – 50 % optical development in the Lenne team

Contribution of the facility • We did : industrial trainee with CNRS , Inserm ( Image J) • Teaching in Master degree, option metrology • Mifobio 2014 : co-organization

Electron Microscopy In the facility, we work as project Project steps - Scientific discussion : How we can help you ? - Technical discussion : Which type of experiment, we choose - Protocol and sample preparation: In collaboration with the team - Imaging : EM and Team people - Conclusion : Help for image interpretation

Electron Microscopy We are a facilities Jean-Paul chauvin Aicha Aouane Fabrice Richard of 3 peoples IE1 TCS IE2 We have projects with IBDM And many equipment TEM CIML SEM Timone Hospital Ultracryomicrotome Inmed AFS => cryossubstitution IMM HPF => cryofixation and much more in or out of PACA and more In 2013 : 33 Projects in 2013 => 42,42 External project Internal project 57,58

Electron Microscopy Facility news : - Training and lecture : The people perform Lecture and training for Labex inform - New equipment : Tecnai G 20 - New technics : Tomography, cryo-imaging, STEM - The facilities always propose training, equipment and project to answer your scientific question Existing equipment New microscope

Electron Microscopy New microscope : Tecnai G20 => Fei Concluding test (4 June) => Facility people training and test the microscope (August) => Finish the existing projects => Open to all people (August) New Technics with the new microscope: => Electron Tomography (3D visualization of EM sample) => Cryo-imaging => STEM Future equipment : => 3D visualization by Scanning electron microscopy (with new microscope)

If you have used the Picsl-FBI imaging core facility, don't forget to thank it: We acknowledge the France- BioImaging/PICsL infrastructure (ANR-10-INSB-04-01).

Imaging Immunity platform @ CIML PICsL / FBI

Service Fusion of 2 platforms Imag ing Imm unity  ImagImm is still a part of PICsL People  Supervisors Didier Marguet & Philippe Pierre  CDD (until Sept. 2014) Sophie Brustlein  IE Mathieu Fallet  IR Sébastien Mailfert

Equipment: 15 microscopes from molecules to organisms 3 Widefield + temperature control Courtesy of M. Sieweke’s lab 3 Confocal 2 Two photon 4 Single molecule Courtesy of M. Dalod’s lab 3 Spinning disk + temperature and + temperature control anesthesia control + temperature control Courtesy of P. Golstein’s lab Courtesy of H&M’s lab Courtesy of M. Bajénoff’s lab

Equipment: 15 microscopes from molecules to organisms 3 Widefield Bleaching low Resolution medium Speed acq. good Multicolor 3 <30µm Depth (z) 3 Confocal Bleaching high Resolution good Speed acq. medium Multicolor 4-8 Depth (z) <30µm 2 Two photon 4 Single molecule 3 Spinning disk Bleaching Bleaching Bleaching low medium low Resolution good Resolution medium Resolution good Speed acq. excellent Speed acq. medium Speed acq . excellent Multicolor 2 Multicolor 2-3 Multicolor 1 Depth (z) <30µm Depth (z) <200 µm Depth (z) <30µm

Trainings (nber of user trained) Significant increase over the last years of the service by CIML’s teams Technique 2013 2014 (January to June) Widefield microscopy NA 40 Confocal imaging NA 50 Spinning disk NA 10 Image Processing NA xxx Use of the platform (100% as for 50h per week) 2013 2014 (January to May) Widefield microscopy 28% 20% ↘ Confocal imaging 78% 87% ↗ (LSM780 + SP5) (92% + 64%) (99% + 75%) Spinning disk 15% (Perkin) 22% ↗ (Perkin + Visitron + polar) (33% + 14% + 20%)

Equipment: what are the needs?  Confocal microscope: LSM780 overbooked (100%) For fixed samples, multi-color imaging (up to 7 colors), high sensitivity  Fluorescence slide scanner High content screening is a clear need to image large number of slides (service from the histology platform)

External trainings  Atelier INSERM - Imagerie du petit animal-imagerie biphotonique - 7 février 2013  EMBO practical courses – Superresolution and single molecule microscopies in living cells – 13/19 mai 2013  PhD program CIML - Cours et TP – 27/29 mai 2013  Atelier de formation INSERM – Cytométrie en flux : du photon à la cellule – 19/22 novembre 2013  Atelier INSERM - De la video-microscopie à la microscopie confocale – 28/30 novembre 2013  PhD program LABEX INFORM - Cours et TP – 13/15 janvier 2014  Atelier de formation INSERM – Cytométrie en flux : du photon à la cellule – 20/24 octobre 2014

R&D in progress CFP-CFP CFP - Tom Intravital multiphoton microscopy (3 colors and CARS/SHG/THG) CFP - YFP Obtain more constrats in living animal imaging Tom - Tom YFP-YFP collaboration with Leica YFP - Tom Y 180 Excitation-polarization-resolved confocal microscopy 160 140 Molecular order in living cell at video rate 120 collaboration with the MOSAIC group at Institut Fresnel 100 80 60 40 20 Super-resolution (STORM/PALM) Molecular distribution and counting collaboration with the PhyTi group at Institut Fresnel Multimodal FCS setup (FCCS/FLIM/STED) Molecular interaction in living cells collaboration with ISS