title: Correlative in vivo 2-photon imaging and focused ion beam scanning electron microscopy: 3D analysis of neuronal ultrastructure.
abstract: This protocol describes how dendrites and axons, imaged in vivo, can subsequently be analyzed in 3D using focused ion beam scanning electron microscopy (FIBSEM). The fluorescent structures are identified after chemical fixation and their position highlighted using the 2-photon laser to burn fiducial marks around the region. Once the section has been stained and resin embedded, a small block is trimmed close to these marks. Serially aligned EM images are acquired through this region, using FIBSEM, and the neurites of interest then reconstructed semi-automatically using the Ilastik software (ilastik.org). This fast and reliable imaging and reconstruction technique avoids the use of specific labels to identify the features of interest in the electron microscope and optimizes their preservation for high-quality imaging and 3D analysis.
authors.affiliation: BioEM Facility, Centre of Electron Microscopy, EPFL, Lausanne, Switzerland; Computer Vision Laboratory, EPFL, Lausanne, Switzerland.
authors.forename: Bohumil
authors.initials: B
authors.lastname: Maco
authors.affiliation: Department of Neuroscience, University Medical Center (CMU), University of Geneva, Geneva, Switzerland.
authors.forename: Anthony
authors.initials: A
authors.lastname: Holtmaat
authors.affiliation: Computer Vision Laboratory, EPFL, Lausanne, Switzerland.
authors.forename: Anne
authors.initials: A
authors.lastname: Jorstad
authors.affiliation: Computer Vision Laboratory, EPFL, Lausanne, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Fua
authors.affiliation: BioEM Facility, Centre of Electron Microscopy, EPFL, Lausanne, Switzerland.
authors.forename: Graham W
authors.initials: GW
authors.lastname: Knott
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology; D057918:Fiducial Markers; D021621:Imaging, Three-Dimensional; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008856:Microscopy, Fluorescence; D008867:Microtomy; D009474:Neurons / Q000648:ultrastructure*; D016613:Plastic Embedding; D013194:Staining and Labeling; D016707:Tissue Fixation
keywords: 2-Photon microscopy; 3D model; 3D reconstruction; CLEM; FIBSEM; Focused ion beam SEM; In vivo imaging; Laser branding; NIRB; NeuroMorph tool; Segmentation; Volumetric analysis