Addressing Particle Compositional Heterogeneities in Super‐Resolution‐Enhanced Live‐Cell Ratiometric pH Sensing with Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticles
Rachel Lee, Jacob A. Erstling, Joshua A. Hinckley, Dana V. Chapman, Ulrich Wiesner
Abstract
The interrogation of metabolic parameters like pH in live-cell experiments using optical super-resolution microscopy (SRM) remains challenging. This is due to a paucity of appropriate metabolic probes enabling live-cell SRM-based sensing. Here we introduce ultrasmall fluorescent core-shell aluminosilicate nanoparticle sensors (FAM-ATTO647N aC' dots) that covalently encapsulate a reference dye (ATTO647N) in the core and a pH-sensing moiety (FAM) in the shell. Only the reference dye exhibits optical blinking enabling live-cell stochastic optical reconstruction microscopy (STORM). Using data from cells incubated for 60 minutes with FAM-ATTO647N aC' dots, pixelated information from total internal reflection fluorescence (TIRF) microscopy-based ratiometric sensing can be combined with that from STORM-based localizations via the blinking reference dye in order to enhance the resolution of ratiometric pH sensor maps beyond the optical diffraction limit. A nearest-neighbor interpolation methodology is developed to quantitatively address particle compositional heterogeneity as determined by separate single-particle fluorescence imaging methods. When combined with STORM-based estimates of the number of particles per vesicle, vesicle size, and vesicular motion as a whole, this analysis provides detailed live-cell spatial and functional information, paving the way to a comprehensive mapping and understanding of the spatiotemporal evolution of nanoparticle processing by cells important, e.g. for applications in nanomedicine.