Lyon Multiscale Imaging Center is a microscopy facility opened to academic research and industry. It federates 3 imaging centers in the Lyon area and provides multi-scale imaging tools including light and electron microscopy:
Machines can be operated independently by the users after appropriate training. Users can also benefit partial or full assistance for their projects. LyMIC provides training sessions for groups or individuals.
The team is watching technology progress and implement new techniques to provide state-of-the-art tools.
We possess unique Brillouin microscopes for imaging different cells and tissues at various scales:
We also possess the expertise to analyze, process and model the Brillouin data to obtain information on the mechanical properties (including anisotropy). Home-made microfluidic devices to mount the samples in special conditions (perfusion, mechanical compression, osmotic shocks…) can be engineered with the input of the Biophysics team. Standard microscopy is also available at the ILM (spinning disk, time lapse…)
Publications
The Liphy R&D team has a long experience in collaboration, joint-development and consulting in optic projects between physics and biology labs. Recently, our team developed a robust method to obtain absolute values of FRET from any epifluorescence microscope. The QuanTI-FRET method calibrates the experimental system and the fluorophore pair, allowing for absolute FRET efficiency and stoichiometry measurements in living cells. Current work focuses on skipping the calibration step with specific samples, hence offering a direct calibration on the sample of interest. This project has been partly funded by the SATT Linksium, an intellectual property has been registered on the software part and discussions are ongoing with private partners.
Another part of the team’s expertise is reflection interference contrast microscopy which allows the measurement of distances with nanometric precision in the vicinity of a reference surface, and to assess the surface functionalization in situ without staining (quantification, quality control). The combination with force application techniques such as flow chambers permits probing biomechanics through the simultaneous control of the force (applied) and the distance (measured). This is essential to study adhesions forces of different organisms: from bacteria to immune cells. The dedicated microscope is fully automated with temperature control, and the PI is currently working on the development of a user-friendly interface for biology-oriented projects.
Both developments are unique in France.
Since 2010, we have developped and implemented numerous methods in optogenetics, and even chemogenetics, in order to have access to dynamics and reversible perturbations of key biological functions such as cell adhesion, cell signaling, transcription factors, inflammation, functions of immune cells and even metabolism. Our lab proposes to share this expertise to the FBI users through access for consulting and even direct collaboration. In the Rhonalpin node, our lab is focused on the coupling between optogenetics, biosensors and metabolism imaging through FLIM imaging. Indeed, we have developed a TIRF microscope presenting a module of FastFLIM imaging. Through the use of dark acceptors, this technology allows the users to extend the possibilities in terms of combining optogenetics, biosensors, metabolic imaging through ratiometric probes and classical multicolors TIRF imaging. Moreover, TIRF imaging allows long term live imaging with low levels of photoxicity. This is essential for metabolic imaging and preserving photon budget for FLIM imaging. In the future, this system will be coupled with a module of evanescent field patterning (EFP) in order to have a specific TIRF-mode illumination of only a region of interest (microm scale).
1- An optogenetic approach to control and monitor inflammasome activation. Julien Nadjar, Sylvain Monnier, Estelle Bastien, Anne-Laure Huber, Christiane Oddou, Léa Bardoulet, Gabriel Ichim, Christophe Vanbelle, Bénédicte Py, Olivier Destaing*, Virginie Petrilli*. Recently accepted in Science Signaling. bioRxiv 2023.07.25.550490; doi: https://doi.org/10.1101/2023.07.25.550490
2-Optogenetic control of YAP cellular localisation and function. Toh PJY, Lai JKH, Hermann A, Destaing O, Sheetz MP, Sudol M, Saunders TE. EMBO Rep. 2022 Sep 5;23(9):e54401.
3-Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying its signaling pathway usage. Kerjouan A, Boyault C, Oddou C, Hiriart-Bryant E, Pezet M, Balland M, Faurobert E, Bonnet I, Coute Y, Fourcade B, Albiges-Rizo C, Destaing O. J Cell Sci. 2021 Jan 25;134(2):jcs254599.
4-β1A integrin is a master regulator of invadosome organization and function. Destaing O, Planus E, Bouvard D, Oddou C, Badowski C, Bossy V, Raducanu A, Fourcade B, Albiges-Rizo C, Block MR. Mol Biol Cell. 2010 Dec;21(23):4108-19.
Over the last ten years, the joint effort of the platform PLATIM and the laboratory of Plant Development and Reproduction (RDP) has developed a strong and recognized expertise in the imaging and quantification of cell mechanical properties in living plant issues. Specific pipelines have been developed to map and quantify:
1. Wall mechanical properties through direct measurements of key parameters such as wall stiffness, adhesion, and resistance to piercing, using AFMs and nano-indenters
2. Cell hydrostatic pressure through stiffness measurements made using AFMs or nano-indenters and the subsequent application of physical models but also through the development of a nano-pressure probe (in progress).
In addition of the originality of these technological approaches in mecanotransduction, the interest of our R&D teams is to develop and support their evolution directly into the environment of an IBiSA core facility. Thus, the R&D Team PLATIM/RDP is one of the rare facilities in France to provide different and integrated mechanical evaluations for both plant and animal multicellular organisms.
Publications:
The development occurring in the platform MEC is tighly associated with joint effort with the “platform of nanocaractérisation” (PFNC) from CEA, leading to have a unique expertise in 3D electron microscopy of large tissue volumes reaching tens of thousands of cubic micrometers at the nanometer scale. This is essential to analyze rare events and true 3D morphological details in neurobiology to study finely cell connectivity, the characterization of membrane structures in chloroplasts from algae and plants, or to study nanoparticles toxicity. Indeed, EM stacks are acquired with much less efforts than by serial sectioning and in a relatively short time (preparation of the sample 3 days, acquisition of the stack 1 day due to high quality stabilization of the samples) new biological questions of interest to the FBI community can be addressed.
Moreover, we have developed a unique pipe-line of analysis of large stacks of 3D EM images. This pipe-line is composed of segmentation steps followed by Ilastik approach for recognition of objects of interests coupled with a serie of home-made plugin specific for quantification. As requested by the network of French electronic microscopists, we are actively collaborating to make available our process to make this pipe-line available for others colleagues and especially the FBI users, as requested by the network of French electronic microscopists. Our work allowed us to develop international collaboration with Andrea Volterra, a world-class leader in the field of FIB-SEM in Neurobiology.
Transcriptomic heterogeneity of cells in organisms over time and space necessitates state-of-the-art technologies to access this variability and its dynamics in situ. Hence, the Spatial-Cell-ID facility aims to enhance the spatial resolution of MERFISH for pinpointing transcripts of specific genes (ranging from a few to thousands) in situ, achieving cellular and subcellular precision over time. To achieve this, we employ a microfluidics device enabling multiple rounds of hybridization (smFISH) synchronized with an advanced 3D STED microscope, providing precise spatial localization of RNA spots with a resolution of 50 nm. This technology is tailored for whole-mount samples across diverse organisms, spanning from mammalian cells to invertebrate systems such as Drosophila and C. elegans. Supported by EquipEx+ funding, Spatial-Cell-ID will be nationally accessible through the LyMIC core facility. Our facility complements commercial systems focused on imaging larger samples at lower 2D resolution. Our bespoke solution not only surpasses current state-of-the-art capabilities but also maintains its position at the forefront of technology, with potential future integration of genomic and spatial proteomic techniques.
Bouchet M., Urdy S., Guan W., Kabir C., Garvis S., Enriquez J. A simple smiFISH pipeline to quantify mRNA at the single-cell level in 3D. (2023). STAR Protocols. Volume 4, Issue 2, 2023, 102316.