Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

Abstract

Fluorescent labeling of cellular substructures is commonly performed using antibody–organic dye conjugates. Organic dyes do not exhibit ideal optical properties in terms of brightness and photostability, however, in particular when it comes to advanced optical super-resolution microscopy (SRM) applications. Here, we demonstrate the efficient conjugation of widely available secondary antibodies and cationic species to ultrasmall (sub-10 nm) fluorescent silica core─poly(ethylene glycol) shell (core–shell) aluminosilicate nanoparticles (aC′ dots) encapsulating different color dyes for specific targeting and high-quality fluorescence imaging of structures of the cytoskeleton (tubulin and actin) and nucleus, respectively. We show that the different color aC′ dots provide enhanced brightness and photostability relative to their parent dyes. As recently discovered, we further demonstrate that they exhibit photo-induced blinking with low ON–OFF duty cycles enabling optical SRM, for example, in the form of stochastic optical reconstruction microscopy (STORM), without the need for complex imaging setups or cocktails. After carefully optimizing Ab–aC′ dot conjugation as well as cell structure labeling protocols in fixed and permeabilized HeLa and MDA-MB-231 cells, we demonstrate three-color STORM and exemplify improved resolution compared to standard antibody–dye conjugates. This work paves the way to next-generation multifunctional optical probes based on ultrasmall silica nanoparticle platforms for advanced applications in bioimaging, nanomedicine, and beyond.