Single molecule localization based optical super-resolution microscopy (SRM) techniques, and in particular stochastic optical reconstruction microscopy (STORM), are powerful imaging tools to resolve structures below the diffraction limit

In quantum materials macroscopic behavior is governed in non-trivial ways by quantum phenomena. This is usually achieved by exquisite control over atomic positions in crystalline solids. Here we demonstrate that the use of disordered glassy materials provides unique opportunities to tailor quantum material properties. By borrowing ideas from single molecule spectroscopy, we isolate single delocalized π-electron dye systems in relatively rigid ultrasmall (<10nm diameter) amorphous silica nanoparticles. We demonstrate that chemically tuning the local amorphous silica environment around the dye over a range of compositions enables exquisite control over dye quantum behavior, leading to efficient probes for photodynamic therapy (PDT) and stochastic optical reconstruction microscopy (STORM). Results suggest that efficient fine-tuning of light-induced quantum behavior mediated via effects like spin-orbit coupling can be effectively achieved by systematically varying averaged local environments in glassy amorphous materials as opposed to tailoring well-defined neighboring atomic lattice positions in crystalline solids. Resulting nanoprobes have required features proven to enable clinical translation.

Multicolor optical super-resolution microscopy (OSRM) describes an emerging set of techniques for the specific labeling of distinct constituents of multicomponent systems with compatible optical probes, elucidating proximity relationships from far-field imaging of diffraction-limited features with nanometer-scale resolution. While such approaches are well established in the study of biological systems, their implementation in materials science has been considerably slower. In large part, this gradual adoption is due to the lack of appropriate OSRM probes that, e.g., by facile mixing or surface modification, enable orthogonal labeling of specific nanostructures in the condensed state, rather than in aqueous conditions as with biology. Here, OSRM probes in the form of ultrasmall (diameters <10 nm) aluminosilicate nanoparticles encapsulating different fluorescent dyes are tailored to visualize both nanodomains of polystyrene-block-poly­[(allyl glycidyl ether)-co-(ethylene oxide)] (PS-b-P­(AGE-co-EO)) diblock copolymer thin films. Careful design of nanoprobe surface chemical properties facilitates either selective compatibilization with the nonpolar PS matrix or preferential reactivity with surface allyl groups of the hydrophilic P­(AGE-co-EO) minority block. Stochastic optical reconstruction microscopy (STORM) of the resulting polymer–inorganic nanocomposite thin films shows nanodomain features of the two chemically dissimilar blocks consistent with atomic force microscopy results. This work paves the way for multiplexed OSRM analysis of polymer nanocomposite bulk structures.

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, 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 are introduced. Only the reference dye exhibits optical blinking enabling live-cell stochastic optical reconstruction microscopy (STORM). Using data from cells incubated for 60 min 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.

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.