title: Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers.
abstract: In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: Martijn T
authors.initials: MT
authors.lastname: Haring
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: Nalan
authors.initials: N
authors.lastname: Liv
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: A Christiaan
authors.initials: AC
authors.lastname: Zonnevylle
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Narvaez
authors.affiliation: Delmic BV, Delft, The Netherlands.
authors.forename: Lenard M
authors.initials: LM
authors.lastname: Voortman
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: Pieter
authors.initials: P
authors.lastname: Kruit
authors.affiliation: Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Hoogenboom