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.

SUBMITTER: Haring MT 

PROVIDER: S-EPMC5333625 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature