Imaging nanoparticle-algae interactions in three dimensions using Cytoviva microscopy

Plasmonic resonances of metal-based nanoparticles are increasingly used for ultrasensitive imaging assays. In this context, the Cytoviva(TM) microscopy platform has greatly gained in popularity. In essence, Cytoviva is an optimized dark field microscope that permits detection of particles down to a few nanometers in size. A significant limitation of Cytoviva up to now has been that it only provided for single plane imaging. The datasets produced by this technique therefore only show a partial view of the sample - not ideally suited to analysis. Here we explain how to overcome this limitation by mounting the Cytoviva condenser on an automated microscope with Z-scanning capability. Our method allows three-dimensional mapping of nanoparticles in their full three-dimensional cellular context. We apply this technique to study the interaction of silver and cerium dioxide nanoparticles with cells of the green alga, Pseudokirchneriella subcapitata, a system of significant environmental relevance because algae underlie much of the aquatic food chain. Our objective was to develop a technique to visualize in detail the interaction of nanoparticles with cells in three dimensions, such that one may, for example, determine whether a particular nanoparticle is inside a cell, at its very surface, or at a distance from it.