Light Microscopy

Equipment

All the equipments are accessible after training by the facility engineers.

AVAILABLE MICROSCOPES

Stand

Nikon Eclipse 80-i microscope

Software

NIS-Elements

Fluorescence lamp

Metal Halide Fluorescent Lamp (130 W, Nikon)

Halogen lamp (Nikon)

Objectives

10x PLAN FLUOR sec (NA: 0.3) (Nikon)

45x PLAN FLUOR dry (NA: 0.75) (Nikon)

60x PLAN APO oil immersion (NA: 1.4) (Nikon)

100x PLAN APO oil immersion (NA: 1.4) (Nikon)

Filter cubes

DAPI cube: BP 402/50, DM LP 409 nm, BP 447/60 (Semrock)

FITC cube: BP 482/36, DM LP 506 nm, BP 536/40 (Semrock)

TRITC Cube: BP 543/22, DM LP 562 nm, BP 593/40 (Semrock)

Camera

Color camera Ds-Ri1, Nikon, (pixel size: 6.45 x 6.45 µm)

Stand

Inverted DMI 6000 microscope (Leica) with motorized x,y,z stage.

Software

MetaMorph version 7.7 (Molecular Devices)

LED sources

CoolLED pE-4000: wide spectrum of LED available from 365 to 770nm

(365, 385, 405, 435, 460, 470, 490, 500, 525, 550, 580, 595, 635, 660, 740, 770 nm)

Halogen lamp for transmission light (Leica)

Objectives

5x sec + DIC (NA: 0.15) (Leica)

10x PLAN FLUOTAR sec + DIC (NA: 0.4) (Leica)

20x PLAN APO multi-immersion + DIC (NA: 0.7) (Leica)

40x APO dry (NA: 0.6) (Leica)

63x PLAN APO oil immersion + DIC (NA: 0.6-1.4) (Leica)

63x PL APO water immersion + DIC (NA: 1.2) (Leica)

Emission Wheel Prior Scientific

55 ms between two adjacent filters

10 positions for 25mm filter

Dichroic Mirrors

Triple band dichroic mirror CFP/YFP/mCherry (69008bs, Chroma)

Quad band dichroic mirror DAPI/FITC/Cy3/Cy5 (89100bs, Chroma)

Emission Filters

Bandpass 455/50 nm (Chroma, F47-456)

Bandpass 470/24 nm (Chroma, F49-469)

Bandpass 525/36 nm (Chroma, F47-526)

Bandpass 535/30 nm (Chroma, F47-535)

Bandpass 632/60 nm (Chroma, F47-632)

Bandpass 700/75 nm (Chroma, F47-700)

Triple bandpass 469/30 – 537/35 – 648/105 (Chroma)

Quad bandpass 447/45 – 520/30 – 598/44 – 704/72 (Chroma)

Camera

Black and white CoolSNAP HQ2 CCD, Photometrics, (pixel size: 6.45 x 6.45 µm)

Color

  • Complementary modules
    • Incubator for stage : Complete T° and CO2 control system (TOKAI HIT)

WSKMX chamber with touch sensitive temperature and CO2 controller (with gas mixer for 100% CO2)

Stand

Nikon Eclipse Ti E inverted microscope, motorized x,y,z stage with Perfect Focus System.

Software

MetaMorph version 7.7 (Molecular Devices)

Objectives

10x PLAN APO sec (NA: 0.45) (Nikon)

40x PLAN FLUOR oil immersion (NA: 1.30) (Nikon)

60x APO TIRF oil immersion (NA: 1.49) (Nikon)

100x PLAN APO oil immersion (NA: 1.40) (Nikon)

100x APO TIRF oil immersion (NA: 1.49) (Nikon)

Filter cubes

DAPI cube: BP 360/40, DM 400 nm, BP 460/50 (Nikon)

BV2A cube: BP 420/40, DM 455 nm, LP 470 (Nikon)

GFP BP cube: BP 470/40, DM 495 nm, BP 520/35 (Nikon)

GFP LP cube: BP 472/30, DM 505 nm, LP 510 (Nikon)

YFP Cube: BP 500/20, DM 515 nm, BP 535/30 (Nikon)

TxRed cube: BP 560/40, DM 595 nm, BP 630/60 (Nikon)

CY5 Cube: BP 620/60, DM 600 nm, BP 700/75 (Nikon)

Lasers

405 nm (Vortran, 100 mW)

488 nm (Vortran, 150 mW)

561 nm (Coherent, 100 mW)

642 mW (Vortran, 100 mW)

Spinning Disk Module

Yokogawa CSU-X1-A1

Dichroic mirrors (Spinning-Disk)

Single band 491 nm (Semrock)

Dual band 491/561 nm (Semrock)

Quad band 405/491/561/641 nm (Semrock)

Spinning Disk 10-position emission filter wheel

Bandpass 447/60 nm (Semrock)

Bandpass 525/45 nm (Semrock)

Bandpass 590/20 nm (Semrock)

Bandpass 607/36 nm (Semrock)

Bandpass 692/40 nm (Semrock)

Quad bandpass 440/40 nm, 521/20 nm, 607/34 nm, 700/45 nm (Semrock)

Longpass 420 nm (Semrock)

Longpass 500 nm (Semrock)

Cameras

Orca Flash 4.0 LT sCMOS, Hamamatsu, (pixel size: 6.45 x 6.45 µm)

Prime 95B sCMOS camera, Photometrics, (pixel size: 11 x 11 µm)

Dual View

Dual View DV2 (Photometrics) :

Dichroic mirror 565 nm (Chroma)

Bandpass GFP (ET525/50m, Chroma) and mCherry (ET630/75m, Chroma) filters.

FRAP/Photoactivation

iLAS2 module (Roper Scientifics)

Super-resolution system

Live SR 3D (Gataca systems)

TIRF

Azimuthal TIRF module iLAS2 (Roper Scientifics)

Dichroic mirrors (TIRF)

Triple band 405/491/561 nm (Semrock)

Quad band 405/491/561/640 nm (Semrock)

10-position emission filter wheel (TIRF)

Bandpass 525/50 nm (Chroma)

Bandpass 605/52 nm (Chroma)

Bandpass 700/75 nm (Chroma)

  • Complementary modules :
    • Super Z stage scanning
    • Thermostatic chamber with CO2/air injection

Stand

Leica DMI 6000 CS inverted microscope

Software

CUDA station equipped with LAS-X software and optional modules:

Dye Finder – Dye Assistant

FRAP – FRET

3D

Lambda Scanning – Excitation/Emission Spectrum

FALCON (Fast Life time CONtrast)

Navigator

Lamps

Metal Halide Fluorescent Lamp (120 W, Leica)

Halogen lamp (Leica)

Lasers

405 nm diode (Leica, 50 mW)

pulsed lasers:

Argon white laser WLL E laser: (470 nm – 670 nm)

Pulsepicker to modulate laser frequency: 80 MHz, 40 MHz, 20 MHz

440 nm pulsed laser 40 MHz

Objectives

10x PLAN APO dry + DIC (NA: 0.4) (Leica)

20x PLAN APO multi-immersion (oil, glycerol, water) + DIC (NA: 0.7) (Leica)

40x PLAN APO oil immersion + DIC (NA: 1.3) (Leica)

63x PLAN APO oil immersion + DIC (NA: 1.4) (Leica)

63x PL APO water immersion + DIC (NA: 1.2) (Leica)

AOBS system

Filter cubes

Cube A: BP 360/40, DM 400 nm, LP 425 (Leica)

Cube I3: BP 460/20, DM 510 nm, LP 515 (Leica)

Cube N3: BP 546/12, DM 565 nm, BP 600/40 (Leica)

Cube Y3: BP 545/30, DM 565 nm, BP 610/75 (Leica)

Detectors

2 PMTs (Hamamatsu 6357)

2 HyD

1 PMT Transmission

  • Complementary modules
    • Super Z stage scanning
    • Thermostatic insert with CO2/air injection

Stand

Leica DM 6000 upright microscope

Software

LAS-X

Module: 3D

Fluorescence lamp

Metal Halide fluorescence lamp (120 W, Leica)

Halogen lamp (Leica)

Lasers

Diode 405 nm (Leica, 50 mW)

Argon laser 458 nm, 476 nm, 488 nm, 496 nm, 514 nm

DPSS laser 561nm

HeNe laser 594 nm, 633nm

Scanning system

Conventional scanner 0.1 – 1.8 kHz (up to 7 frames/sec in 512×512)

Resonant scanner 8 kHz (up to 30 frames/sec in 512×512)

Objectives

20x PLAN APO dry (NA: 0.75) (Leica)

63x PLAN APO oil immersion + DIC (NA: 1.4) (Leica)

AOBS system

Filter cubes

Cube A: BP 360/40, DM 400 nm, LP 425 (Leica)

Cube I3: BP 460/20, DM 510 nm, LP 515 (Leica)

Cube N3: BP 546/12, DM 565 nm, BP 600/40 (Leica)

Detectors

1 PMT (Hamamatsu 6357)

2 GaAsP Hybrid (Hamamatsu)

1 PMT Transmission

Stand

Nikon AZ100 MultiZoom macroscope

Software

NIS-Elements D (Nikon) + Extended Depth of Focus

Lamps

Metal halide fluorescent lamp (130 W Intensilight, Nikon)

Halogen lamp (Nikon)

Ring light

Objectives

AZ-Plan Apo 1X NA 0.1 WD 35 mm (Nikon) + DIC

AZ-Plan Fluor 2X NA 0.2 WD 45 mm (Nikon)

AZ-Plan Fluor 5X NA 0.5 WD 15 mm (Nikon) + DIC

Filter cubes

A0 cube: BP 340/26, DM LP380 nm, LP364 RS (Semrock)

DAPI cube: BP 377/50, DM 409 nm, BP 447/60 (Semrock)

CFP-2432A cube: BP 438/24, DM 458 nm, BP 483/32 (Semrock)

GFP-BP cube: BP 472/30, DM 495 nm, BP 520/35 (Nikon)

GFP-LP Cube: BP 480/40, DM 505 nm, LP520 (Nikon)

YFP-2427A cube: BP 500/24, DM 520 nm, BP 542/13 (Semrock)

TRITC-A cube: BP 543/22, DM 562 nm, BP 593/40 (Semrock)

Camera

Ds-Ri color camera, Nikon, (pixel size: 6.45 x 6.45 µm)

MultiZoom

Zoom 1X to 8X

Stand

Nikon Eclipse Ti E inverted microscope, motorized x,y,z stage with Perfect Focus System.

Software

NIS-Elements Advanced Research

Fluorescence lamp

Metal Halide fluorescence lamp (130 W, Nikon)

Halogen lamp (Nikon)

Lasers

405 nm diode (Cube, 100 mW, Coherent)

488 nm (Sapphir, 150 mW, Coherent)

561 nm (Sapphir, 150 mW, Coherent)

647 nm (MPB Communication, 300 mW)

Objectives

20x PL APO sec (NA: 0.75) (Nikon)

100x APO TIRF SR oil immersion (NA: 1.49) (Nikon)

Filter cubes

DAPI cube: BP 377/50, DM 409 nm, BP 430/45 (Nikon, Semrock 2SKZ4DAPIB)

FITC cube: BP 482/35, DM 506 nm, BP536/40 (Nikon, Semrock 2SZK4FITC0)

TRITC Cube: BP 543/22, DM 562 nm, BP 593/40 (Nikon, Semrock 2SKZ4TRITC)

Cube QUAD: Compatible for laser 405/488/561/647 (n°241578), DM 405/488/561/647 (n°241164), 450/60-525/50-605/50-730/120 (n°241102) (Nikon, Chroma)

Camera

Andor iXon Ultra 897 EM-CCD (pixel size: 16×16 µm)

IMAGE ANALYSIS STATIONS AND SOFTWARES

Manufacturer – Model

Dell Inc.

Precision WorkStation T5500

Windows 10 (64bit) & Linux Ubuntu

Components-Processor

Intel(R) Xeon(R) Gold 6140 2.3G

Memory/RAM

256 Giga DDR4

Graphics Card

Nvidia Quadro P600 (24GB)

Softwares

Huygens Essential Deconvolution (SVI)

NIS-Elements Advanced Research

Leica LAS X

Metamorph

Matlab

Chimera

Fiji / ImageJ

Icy

Manufacturer – Model

Dell Inc.

Precision Tower 7810

Windows 10

Components-Processor

Intel(R) Xeon(R)

CPU E5-2640 v3 @ 2.60GHz

Memory/RAM

128GB RIMM

Graphics Card

Nvidia Quadro M4000

Softwares

Imaris 9.1 Advanced Tracking

modules: MeasurementPro, ImarisTrackLineage, ImarisColoc, and

ImarisVantage.

Leica LAS X core offline

Metamorph

Matlab

Chimera

Fiji / ImageJ

Icy

3D PRINTER

Modeling by deposition of molten material (FDM)

2 printing nozzles

Types of materials: PLA, PVA, ABS, …

Protective cover

Production of parts

Rates

Training

Training part soon

Services

Services soon

Conditions of Use

Charter of the Imaging-Gif platforms

  1. Daily hours, laboratory openings, access to the facility.

Users can choose their arrival time within the 9:00 am – 6:00 pm range.
Access to the I2BC outside these times may be expressly authorized by the technical manager of the platform.
Users must respect the health and safety instructions posted in the facility at their disposal (see also paragraph 3.4 Isolated work of the present charter).
The Platforms operate throughout the calendar year. A table of engineers’ absences is clearly displayed; users must take these absences into account in the case of scheduling accompanied work. The periods of closure are decided by the scientific director of Imagerie-Gif.

  1. Diffusion of scientific results
    2.1. Confidentiality

Everyone is required to respect the confidentiality of the work entrusted to them and their colleagues. In case of disclosure of information (publications, patents, communications based on the use of Platform resources), it is strongly recommended to submit the Materials and Methods/Results parts obtained on the Platform equipment to the technical manager of the Platform. The results and data obtained from researches conducted on the Platform are not the property of the Platform but are subject to the regulations of the various Institutions covering each user. On the other hand, the originals of the laboratory notebooks of the engineers involved in the support must be kept on the Platforms.
Platform engineers are authorized to use the results acquired on the Platform in their actions of valorization of the platform under cover of authorization of the project authors.

2.2. Publications

Publications involving results obtained on one of the Imaging-Gif Platforms must specify this in the acknowledgements. If the engineers appear as co-authors, their belonging to the unit and their affiliation to the supervisors must appear in the address.

A copy of all publications (articles, journals, theses, …) for which all or part of the work was carried out within the platforms must be given to the technical manager of the Platform as soon as they are published.

  1. Health and safety
    3.1 Safety

Platform staff must ensure that user agents have received safety training and, if necessary, specific training adapted to their work on the Platform. If they have not, they undertake to provide them with such training.

3.2 No smoking

Smoking is prohibited in the workplace (Decree 92-478 of May 29, 1992).

3.3. Lone working

Lone working must be exceptional and dedicated to tasks that do not present a risk. In the event that dangerous work must necessarily be performed outside of official opening hours, it is mandatory to be accompanied. The line manager and the technical manager of the Platform must be notified.

3.4. GMOs: Specific measures

The use of GMOs (Genetically Modified Organisms) is regulated. The official information on their use can be consulted on the CNRS sites.

The main rules for the handling of GMOs at I2BC are the following:

– All GMOs must be handled, stored, decontaminated, and disposed in a manner that:
1. Do not harm the health of the users
2. Avoid contamination of other users biological material
3. Avoiding dissemination

– GMOs include microorganisms (bacteria, viruses, yeast, etc, …), plants and transgenic cell cultures

– Before disposal, all GMOs must be sterilized by bleach treatment or autoclaving

– Transgenic plants must be grown and handled in greenhouses or enclosures according to the rules available in these places and on the Intranet

– The level of containment (L1, S1, S2 and S3) required for each type of experiment has been notified for each project by the « Commission du Génie Génétique » (CGG). This information can be consulted in the application files for approval of contained use of genetically modified organisms (groups I and II) filed with the I2BC secretariat and can be consulted on the Intranet

– Any new type of experiment must be the subject of a request for modification of the approval of contained use of GMOs application.

3.5. Radioactivity : Specific measures

Using radioactive markings must be reported to the technical manager of the platform at first appointment.

4 – Training

The engineers inform and advise users on their training needs and requests. They will eventually set up specific and adequate trainings to the expressed needs.

5 – Use of computer resources

Each user of the Platforms hosted at the I2BC and using the I2BC’s computer resources must take note of the I2BC’s computer charter.

It is strictly forbidden to use the computers dedicated to the control of the Platform equipment for purposes other than the acquisition or analysis of data related to the equipment (prohibition of internet or intranet consultation, e-mail, installation of programs, etc.).

6 – Use of collective technical resources

Engineers are responsible for the maintenance of equipment and laboratories. However, it is essential that the conditions and rules of use are rigorously applied by each user. The Platforms engineers must ensure that users are trained and informed of the safety rules. They reserve the right to deny access to the Platform in case of non-compliance with the rules of behavior.

Contact

Plateforme de Microscopie Photonique / Light Microscopy Facility
Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC)
1 Rue de la Terrasse

91198, Gif-sur-Yvette, France.

 

Le Bars Romain, Head of facility, Researcher Engineer

Mail : romain.lebars@i2bc.paris-saclay.fr

Phone: +33 1 69 82 46 40

 

Lecart Sandrine, Researcher Engineer

Mail : sandrine.lecart@i2bc.paris-saclay.fr

Phone: +33 1 69 82 46 40

 

Temagoult Mébarek, Engineer

Mail : mebarek.temagoult@i2bc.paris-saclay.fr

Phone: +33 1 69 82 46 40

 

Acknowledgments

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