Published: 2023-10-25
Our next Special Edition Virtual Pub, “Multiscale Imaging in the Neurosciences,” organized in collaboration with EBRAINS, will take place on Friday, October 27. At this event, we will explore the potential areas of collaboration between EBRAINS and Euro-BioImaging and highlight the expertise of selected Euro-BioImaging Nodes in the Neuroscience domain.
When: October 27, 2023, from 13:00-15:00 CEST
Where: Online
At this event, Mathieu Ducros of France BioImaging explains the potential of Lattice Lightsheet Microscopy for studying live neuronal tissue. Join us on Friday, October 27, to hear this talk and others like it.
Abstract
Super-resolution imaging inside brain slices with lattice light-sheet microscopy
Mathieu Ducros, France BioImaging
Lattice light sheet microscopy (LLSM)1 combines high spatial resolution, fast 3D imaging and very low photo-toxicity. LLSM is therefore a method with great potential for studying live neuronal tissue. In the past, we have demonstrated the performances of LLSM to image and photo-manipulate organotypic brain slices2,3. However, our imaging resolution was limited by diffraction to ~270 nm. To reach nanometric resolutions, we decided to apply Single Molecule Localization Microscopy (SMLM) modality to LLSM. However, a major challenge to perform SMLM inside a thick sample comes from the optical aberrations, which appear in depth of most biological tissues and degrade image intensity, resolution and thus, localization precision. Here we present a method that combines LLSM, adaptive optics (AO) - a method to correct optical aberration - and DNA paint as the SMLM protocol, and applied it to image structures at super resolution inside organotypic brain slices. We have previously demonstrated an active image optimization (AIO) method, based on light-sheet refocussing and sample’s optical aberrations correction with AO, that showed an intensity and resolution increase in diffraction limited regime4. To demonstrate the performance of our method we imaged GFP expressing neurons in a fixed brain slice. DNA-PAINT was achieved thanks to the sdAB anti GFP kit (Massive Photonics). We successfully observed and reconstructed neuronal structures at super resolution from the surface down to 40µm inside fixed brain slices. Thanks to our AO optimization protocol, we increased the single molecule detection number and decreased the localization uncertainty. LLSM active image optimization method for SMLM inside thick samples opens new perspectives for imaging at the molecular level in an integrated neuronal tissue. Furthermore, this method could be applied to single particle tracking in live tissue. 1. Chen, B. C. et al. Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science 346, (2014). 2. Ducros, M. et al. Lattice light sheet microscopy and photo-stimulation in brain slices. in SPIE Proceedings (2019). 3. Getz, A. M. et al. High-resolution imaging and manipulation of endogeneous AMPA receptor surface mobility during synaptic plasticity and learning. Sci. Adv. 8, (2022). 4. Malivert, M. et al. Active Image Optimization for Lattice Light Sheet Microscopy in Thick Samples. Biomed. Opt. Express (2022).