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PresentationThe 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) a flow-cytometer (VYB), and a cell sorter (TYTO) all enable to work with living cells. 1 - The confocal system has been updated in May 2022 (S-M4D ; Olympus GATACA and Andor). It is based on a motorized IX81 Olympus microscope and equipped with a Nipkow disk (Yogokawa CSU-X1), 6 solid state lasers for 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 - Installed at EMBL (room 158), the TYTO sorter (Miltenyi) has the particularity of being able to sort at low pressure, limiting dilution of the cells, which is important to keep cells alive, particularly for fragile populations. The sorting cartridge contains all the microfluidics required for autonomous and perfectly sterile sorting. Combined with a Dispencell (Seed Bioscience), the sorted cultures can be cloned, paving the way for single-cell applications. 5 - 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 6 - 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 Micro-Manager v2.0 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 (551). Key wordsMicroscopy ; DIC ; Epifluorescence ; 4D ; Flow-cytometry ; Cell sorting ; Spinning-disk confocal ; FRAP ; FRET ; Super-resolution microscopy ; PALM ; STORM ; PAINT ; sptPALM Dedicated StaffThe technical and scientific expertise and the practical management of the platform are under the responsibility of RL Revel-Goyet, M. Pelosse (EMBL), O. Glushonkov and JP Kleman. AccessibilityAuthorized 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. How to make a request ?Requests are made through the online form or by direct contact. Contact and localisationThe platform is located room 547 in the IBS ; the cell sorter (TYTO), at EMBL room 158. SamplesThe 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). ResultsIn 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. CostUsers are charged for the operation costs calculated per hour . Contact us for further information. Follow-up/ValuationThe 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 : |
