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

PICsl-FBI photonic- electronic Imaging Facility

IBDM Imaging facility

Technical supervisor

Cédric Matthews IR2 CNRS

Quality process Video-microscopy Tcn CNRS

Service 11 video-microscopes

Confocal Metrology

service 4 confocal, 3 spinning disk

machining/ microscopy AI CNRS

project computer-aided design / machining Two photon microscopy development Cédric Matthews Service / Project 2 two photon Image processing

scientific supervisor

Pierre-François Lenne DR2 CNRS

1 two photon , 1macro- confocal

200 researchers + 20 industrials + 4 Master Degree’s Training Operating Budget 2014 100 000 euros Capital Budget since 2009 1 million euros

1 IR , 1 AI , 1 TCN 22 systems

Main Equipments

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

New Equipments

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

Available Techniques

systems

• Videomicroscopy
• Confocal
• Two Photon
• Macro confocal
• STED

dynamic imaging and contrast

• FRET
• FRAP
• FCS
• Absorption
• Fluorescence
• SHG
• Depletable dyes

processing and images analysis

• ImageJ- IcY
• Automatisation
• f analysis
• Matlab
• Huygens

deconvolution Prototype

• Turning machine
• Milling
• 3D printing

(coming soon)

Our Expertise

Associated to systems

• Image processing
• Metrology
• Quality control (dyes)
• Master's degree tutoring
• Competitive Bidding

publication

• computer-aided design /

machining

IBDM training

• 5 different trainings :
• Image analysis (ImageJ)
• Macro –development
• confocal Microscopy
• two photon Microscopy
• High resolution

Vocational training

• CNRS / INSERM
• CNRS - company
• Image analysis (ImageJ)
• Imaging and quality

control

4280 hours of use from January to June 2014

Distribution of systems use

videomicroscope confocal two photons STED

Distribution of system trainings

11 individual trainings on video-microscopes and 15 on other microscopes from January to June

spinning LSM 780 LSM 510 video STED

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

In 2013 : 33 Projects in 2013 =>

57,58 42,42 External project Internal project

Electron Microscopy

We are a facilities

• f 3 peoples

Jean-Paul chauvin Aicha Aouane Fabrice Richard 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

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 New microscope Existing equipment

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  ImagImm is still a part of PICsL People

Didier Marguet & Philippe Pierre  Supervisors Sophie Brustlein  CDD (until Sept. 2014) Mathieu Fallet  IE Sébastien Mailfert  IR

Imaging Immunity

2 Two photon

+ temperature and anesthesia control Courtesy of M. Bajénoff’s lab

3 Widefield

+ temperature control Courtesy of M. Sieweke’s lab

3 Confocal

Courtesy of M. Dalod’s lab

3 Spinning disk

+ temperature control Courtesy of P. Golstein’s lab

4 Single molecule

+ temperature control Courtesy of H&M’s lab

Equipment: 15 microscopes

from molecules to organisms

4 Single molecule

Bleaching Resolution Speed acq. Multicolor Depth (z) <30µm 1 low

3 Spinning disk

Bleaching Resolution Speed acq. Multicolor Depth (z) <30µm 2 excellent

3 Confocal

Bleaching Resolution Speed acq. Multicolor Depth (z) <30µm 4-8 high good good low medium Bleaching Resolution Speed acq. Multicolor Depth (z)

3 Widefield

<30µm 3 good medium Bleaching Resolution Speed acq. Multicolor Depth (z) <200 µm 2-3 medium

2 Two photon

medium medium good low excellent

Equipment: 15 microscopes

from molecules to organisms

Use of the platform (100% as for 50h per week)

2013 2014 (January to May) Widefield microscopy 28% 20%↘ Confocal imaging (LSM780 + SP5) 78% (92% + 64%) 87%↗ (99% + 75%) Spinning disk (Perkin + Visitron + polar) 15% (Perkin) 22%↗ (33% + 14% + 20%)

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

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

Intravital multiphoton microscopy (3 colors and CARS/SHG/THG)

Obtain more constrats in living animal imaging collaboration with Leica

Excitation-polarization-resolved confocal microscopy

Molecular order in living cell at video rate

collaboration with the MOSAIC group at Institut Fresnel

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

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