Abstract
Fiducial markers are used in correlated light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. Currently used fiducial markers, e.g. dye-labelled nanoparticles and quantum dots, suffer from irreversible quenching of the luminescence after electron beam exposure. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can (partially) withstand electron bombardment, are interesting because of the recent development of integrated CLEM microscopes. In addition, nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow switching back from EM to LM and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; 130 nm gold-core rhodamine B-labelled silica particles, 15 nm CdSe/CdS/ZnS core-shell-shell quantum dots (QDs) and 230 nm Y(2) O(3) :Eu(3+) particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The gold-core rhodamine B-labelled silica NPs and QDs are quenched after a single exposure to 60 ke(-) nm(-2) with an energy of 120 keV, while Y(2) O(3) :Eu(3+) NPs are robust and still show luminescence after five doses of 60 ke(-) nm(-2) . In addition, the luminescence intensity of Y(2) O(3) :Eu(3+) NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that Y(2) O(3) :Eu(3+) NPs are promising as robust fiducial marker in CLEM. LAY DESCRIPTION: Luminescent particles are used as fiducial markers in correlative light and electron microscopy (CLEM) to enable accurate overlaying of fluorescence and electron microscopy images. The currently used fiducial markers, e.g. dyes and quantum dots, loose their luminescence after exposure to the electron beam of the electron microscope. This limits their use in CLEM, since samples have to be studied with light microscopy before the sample can be studied with electron microscopy. Robust fiducial markers, i.e. luminescent labels that can withstand electron exposure, are interesting because of recent developments in integrated CLEM microscopes. Also nonintegrated CLEM setups may benefit from such fiducial markers. Such markers would allow for switching back to fluorescence imaging after the recording of electron microscopy imaging and are not available yet. Here, we investigate the robustness of various luminescent nanoparticles (NPs) that have good contrast in electron microscopy; dye-labelled silica particles, quantum dots and lanthanide-doped inorganic particles. Robustness is studied by measuring the luminescence of (single) NPs after various cycles of electron beam exposure. The dye-labelled silica NPs and QDs are quenched after a single exposure to 60 ke(-) nm(-2) with an energy of 120 keV, while lanthanide-doped inorganic NPs are robust and still show luminescence after five doses of 60 ke(-) nm(-2) . In addition, the luminescence intensity of lanthanide-doped inorganic NPs is investigated as function of electron dose for various electron fluxes. The luminescence intensity initially drops to a constant value well above the single particle detection limit. The intensity loss does not depend on the electron flux, but on the total electron dose. The results indicate that lanthanide-doped NPs are promising as robust fiducial marker in CLEM.