Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

Haring, M.T. (TU Delft ImPhys/Charged Particle Optics)
Liv Hamarat, N. (TU Delft ImPhys/Charged Particle Optics)
Zonnevylle, A.C. (TU Delft ImPhys/Charged Particle Optics)
Narvaez Gonzalez, A.C. (TU Delft ImPhys/Charged Particle Optics)
Voortman, L.M. (Delmic)
Kruit, P. (TU Delft ImPhys/Charged Particle Optics)
Hoogenboom, J.P. (TU Delft ImPhys/Charged Particle Optics)

2017-03-02

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.

Fluorescence imaging
Microscopy
OA-Fund TU Delft

http://resolver.tudelft.nl/uuid:b0bbecda-c6e6-45e4-925b-d2b3bcd23f5a

https://doi.org/10.1038/srep43621

2045-2322

Scientific Reports, 7

Institutional Repository

journal article

© 2017 M.T. Haring, N. Liv Hamarat, A.C. Zonnevylle, A.C. Narvaez Gonzalez, L.M. Voortman, P. Kruit, J.P. Hoogenboom