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Integrated Structural Biology Grenoble
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Contact person(s) related to this article / GLUSHONKOV Oleksandr / INDORATO Rose-Laure / KLEMAN Jean-Philippe / LACROIX Françoise

Cellular Imaging

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Presentation

The Cellular Imaging platform grants the access to a confocal microscope (spinning disk S-M4D), a video-microscope (V-M4D), two super-resolution setups (SR-M4D and PALM-M4D) and a flow-cytometer (VYB), all enable to work with living cells.

1 - The confocal system (S-M4D; Olympus and Andor) is based on the motorized IX81 Olympus microscope and equipped with a Nipkow wheel (Yogokawa CSU-X1), 6 solid state lasers for a real-time confocal imaging of living cells (low laser phototoxicity and opaque thermostatic enclosure) and a photoconversion/ photoactivation module (used, for example, in FRAP experiments). Images can be acquired simultaneously with 2 EMCCD cameras (Andor iXon ultra). The confocal imaging has few advantages over the traditional epifluorescence technique, including an improved resolution, a higher contrast due to drastic reduction of the background and an optical sectioning.

2 - The epifluorescence imaging station (V-M4D, Olympus) is composed of a new inverted Olympus IX83 fully motorized microscope, a 16-channel LED source, and equipped with a thermostatic incubation chamber for live cell imaging. Acquisition of images is achieved with a sCMOS camera (Hamamtsu Orca Flash4; Volocity)

3 - The flow-cytometer (VYB, Miltenyi biotech) is equipped with three lasers (8 fluorescence channels) and covers the emission wavelengths used on optical microscopes. It is automatable (multiwell plates, immuno-staining, dilutions), and compatible with magnetic columns Miltenyi (isolation of rare events).

4 - The SAFe360 microscope from Abbelight and Olympus (SR-M4D). This new microscope was installed on the M4D imaging platform in 2021 and aims to reinforce our super-resolution capabilities (PALM/ STORM/ PAINT and single particle tracking). The microscope allows 2D/3D multicolor imaging of fixed and living cells with a resolution of few tens of nanometers, as well as single particle tracking experiments with a localization precision up to 5-10 nm for bright organic fluorophores. The system is equipped with a laser combiner (405 nm, 488 nm, 532 nm, 561 nm, 640 nm et 730 nm), a module for homogeneous EPI/HiLo/TIRF illumination and two sCMOS Hamamatsu Fusion cameras offering a large field of view and high acquisition speeds (more than 100 frames per second). The microscope is highly automated and controlled via the NEO software. For a more detailed information you can contact us or visit the manufacturer’s website: https://www.abbelight.com/solutions/abbelight-instruments/#SAFe360

5 - The home-built super-resolution setup (PALM-M4D). This is a noncommercial system based on an inverted Olympus IX81 microscope, equipped with 2 EMCCD cameras (Photometrics) and 6 lasers (405 nm, 488 nm, 532 nm, 561 nm, 643 nm and 730 nm) controlled by an acousto-optic filter (AOTF). The Metamorph software is used to control the microscope with cameras and to perform the acquisitions of images, whereas the Labview code allows a precise lasers control. This microscope is recommended for more experienced users and can be modified/adapted on demand for a particular need or a scientific project. More details on the IBS web site.

Three workstations with the software for microscopy and cytometry data analysis are available in a dedicated room (568).
Image analysis: Volocity and Imaris;
Super-resolution data analysis: NEO and open source solutions (e.g. FiJi);
Cytometric analysis: MacsQuant and Flowlogic.

Key words

Microscopy; DIC; Epifluorescence; 4D, Flow-cytometry ; Spinning-disk confocal; FRAP; FRET; Super-resolution microscopy; PALM; STORM; PAINT; sptPALM

Dedicated Staff

The technical and scientific expertise and the practical management of the platform are under the responsibility of RL Revel-Goyet, F. Lacroix, O. Glushonkov and JP Kleman.

Accessibility

Authorized users can access freely the equipment of the platform (microscope and post-treatment dedicated computer), after they have validated the internal training session, and accepted the conditions of use.
For more complex projects or if a specific expertise is requested (collaboration), we can provide a technical assistance for some or all of the experiments planned (observation and post-treatment).

How to make a request?

Requests are made through the online form or by direct contact.

Contact and localisation

The platform is located room 547 in the IBS.

Samples

The microscope is dedicated to fixed or living Eukaryote and Procaryote cells observation (level 2). Security training is mandatory to access to the room. A tissue culture room is available for customers (L2).

Results

In general, the acquisition data and the analyzed data are stored for 1 year. A spare copy is automatically generated, but the platform cannot guarantee the integrity of the data stored in case of dysfunction. The user is responsible for the final storage of his own datasets. After one year, the data can be erased by the platform supervisors without notification.

Cost

Users are charged for the operation costs calculated per hour . Contact us for further information.

Follow-up/Valuation

The platform can help users who need technical advises (preparation of samples) or during treatment and analysis of the data.

Users should cite the platform in their publications :
« We thank Rose-Laure Revel-Goyet, Françoise Lacroix, Oleksandr Glushonkov and Jean-Philippe Kleman (Institut de Biologie Structurale, Grenoble) for the support and access to the Cell imaging Platform. » and thank FRISBI et GRAL for their UAR support :"This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003)."
For collaborative work, supervisors of the platform must co-sign papers.