Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

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Abstract
In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.

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