Towards robust and versatile single nanoparticle fiducial markers for correlative light and electron microscopy

Abstract

SummaryFiducial 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 Y2O3:Eu3+ 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 Y2O3:Eu3+ NPs are robust and still show luminescence after five doses of 60 ke− nm–2. In addition, the luminescence intensity of Y2O3:Eu3+ 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 Y2O3:Eu3+ NPs are promising as robust fiducial marker in CLEM.Lay DescriptionLuminescent 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‐labelle...

Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

“…CELLocate); printing of coverslips with gold nanoparticles (Benedetti et al, 2014;Prabhakar et al, 2018) or titanium (Rodighiero et al, 2015). It can also involve applying the following onto specimens: single-or multi-color fluorescent beads (Schorb et al, 2017;Watanabe et al, 2014;Watanabe et al, 2011); gold particles (Watanabe et al, 2014); fluorescently labelled gold particles (Mohammadian et al, 2019); quantum dots and Y2O3:Eu 3+ nanoparticles (van Hest et al, 2019).…”

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

“…The well-established fluorescence microscopy markers, including organic fluorophores, fluorescent proteins, and semiconductor quantum dots, are not suitable for CCLEM since their emission stability and brightness are severely limited. 61,79 Organic molecules bleach rapidly under accelerated electrons due to structural damage introduced by the primary beam. 74,79,80 Semiconductor quantum dots (QDs), such as CdSe/ZnS structures, which have gained increasing importance as probes in traditional fluorescence microscopy, suffer from electron ionization, followed by trapping of the ejected electrons at deep traps inside their matrix.…”

“…61,79 Organic molecules bleach rapidly under accelerated electrons due to structural damage introduced by the primary beam. 74,79,80 Semiconductor quantum dots (QDs), such as CdSe/ZnS structures, which have gained increasing importance as probes in traditional fluorescence microscopy, suffer from electron ionization, followed by trapping of the ejected electrons at deep traps inside their matrix. 79,81 As alternatives, rare-earth element (REE) doped nanoparticles 59,64,82 (Fig.…”

“…74,79,80 Semiconductor quantum dots (QDs), such as CdSe/ZnS structures, which have gained increasing importance as probes in traditional fluorescence microscopy, suffer from electron ionization, followed by trapping of the ejected electrons at deep traps inside their matrix. 79,81 As alternatives, rare-earth element (REE) doped nanoparticles 59,64,82 (Fig. 3c) and fluorescent nanodiamonds (FNDs) 65,76,83 (Fig.…”

“…62 However, decreasing CL intensity using low acceleration voltages has been reported. 59,61 Loss in emission intensity in REE 3+ doped nanocrystals can be attributed to knock-on damage 79,84 and the accumulation of active quenchers. 61,62,76 Nonetheless, the stability of inorganic nanocrystals is still significantly higher compared to widely adopted fluorescence microscopy labels.…”