Venue: CARF – Salle Nord
Lydia DANGLOT
Institute of Psychiatry & Neuroscience of Paris,
INSERM U1266, Université Paris Cité, 102 rue de la Santé, Paris, France.
Invited by Etienne Herzog
Title
Light up neuronal architecture and synapse : from 3D STED imaging on thick tissue to single molecule imaging using new Photoswitchable Fluorescent Probes for Live SMLM Imaging.
Abstract
The brain comprises billions of densely packed neurons. Isolating single cells in their native arrangement within brain tissue represents a crucial step for describing neuronal shape, size and complexity, which enables in turn the characterization of cell types and the identification of any morphological abnormalities characterizing neuropathies. To analyze neuronal morphometry within brain tissue, we developped recently, a new pipeline based on 3D-STED microscopy on cleared thick brain slices and new segmentation algorithm. This open software SENPAÏ allows reconstructing the full dendritic arbor and dendritic spine morphology within very dense tissue. We will showcase how it can isolate neuronal morphology on basic transgenic mice expressing GFP in a population of neighbouring Purkinge Cells (Cauzzo et al, Nature Comm 2024[1]).
Elucidating molecular organization in cell biology requires to precisely localize single or aggregated molecules and to analyze quantitatively their spatial distributions. We previously developed a statistical method SODA (Statistical Object Distance Analysis plugin, Nature Comm 2018 ([2]) that uses either micro- or nanoscopy to significantly improve standard co-localization techniques. Our method considers cellular geometry and densities of molecules to provide statistical maps of isolated and associated (coupled) molecules. SODA can be used to detect indirect molecular association in conventional microscopy or even in super resolution microscopy (SIM, STED or STORM) where higher resolution prevents the use of conventional overlay methods. We will show how SODA method can be used to decipher either contact site between ER & plasma membrane in STED [3] or to detect the alignment of synaptic proteins in STORM.
We previously presented the MemBright family [4] which are six fluorescent probes compatible with long-term live-cell imaging (without any use of transfection or transgenic animals) that can be used in 3D multicolor dSTORM in combination with immunostaining [4]. We are now extending MemBright capabilities to Live STORM imaging that requires probes able to spontaneously blink. Those photoconverters [5,6] can in conventional or SMLM imaging to image lipid droplets, plasma membrane, or mitochondria on live samples [6].
[1] S. Cauzzo et al. ” A modular framework for multi-scale tissue imaging and neuronal segmentation”, Nature Commun, volume 15, Article number: 4102 (2024).
[2] T. Lagache et al., “Mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics,” Nat Commun 9(1), 698 (2018).
[3] A. Gallo et al., “Role of the Sec22b-E-Syt complex in neurite growth and ramification,” J Cell Sci 133(18), (2020).
[4] M. Collot et al., “MemBright: A Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience,” Cell Chem Biol 26(4), 600-614 e607 (2019).
[5] L. Saladin et al., “Dual-Color Photoconvertible Fluorescent Probes Based on Directed Photooxidation Induced Conversion for Bioimaging,” Angew Chem Int Ed Engl 62(4), e202215085 (2023).
[6] L. Saladin et al., “Targeted Photoconvertible BODIPYs Based on Directed Photooxidation Induced Conversion for Applications in Photoconversion and Live Super Resolution Imaging,” JACS (2023).