Addressing Particle Compositional Heterogeneities in Super‐Resolution‐Enhanced Live‐Cell Ratiometric pH Sensing with Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticles

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, 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.

Cellulose Nanofiber/Polyvinyl Alcohol-Based pH-Responsive Films Containing Anthocyanin and Carbon Dots

“…4,5 However, compared with the abovementioned methods, colorimetric and fluorescent sensing materials have aroused much interest because of their rapid response, excellent stability, cost effectiveness, and simple operation. 6,7 pH-responsive colorimetric films produce different colors as the ambient pH changes. Generally, pH colorimetric indicators are obtained by mixing dyes sensitive to pH change into a solid matrix.…”

Cellulose-Based Fluorescent Material for Extreme pH Sensing and Smart Printing Applications

“…Until now, several fluorescence pH sensors have been prepared including small organic molecules, covalent organic frameworks, , carbon quantum dots, , nanoparticles, , and fluorescent proteins . However, the preparation of optical sensors for extreme acidic and alkaline pH measurements in a narrow pH range remains challenging, and their availability is still limited.…”

“…Typically, the carboxyl, hydroxyl, and aromatic amines and other nitrogen-containing heterocycles are highly sensitive for acid−base equilibrium processes and have been widely utilized to construct pH probes. 21 Until now, several fluorescence pH sensors have been prepared including small organic molecules, 13 covalent organic frameworks, 22,23 carbon quantum dots, 24,25 nanoparticles, 26,27 and fluorescent proteins. 28 However, the preparation of optical sensors for extreme acidic and alkaline pH measurements in a narrow pH range remains challenging, and their availability is still limited.…”

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

“…20,21 Finally, conjugating a sensor dye to the PEGshell of aC′ dots has subsequently been demonstrated to allow SRM-enhanced ratiometric sensing of metabolic parameters within cells, adding another powerful functionality to these multifunctional optical SRM probes. 22 In order to add highly specific (e.g., intracellular) targeting and immunofluorescent labeling capabilities to this platform, there is a clear need to develop robust protocols allowing efficient conjugation of Abs to the aC′ dot surface. Efforts along these lines are described herein, together with examples of highly effective labeling of specific intracellular structures, and their imaging using STORM.…”

“…They overcome a number of the optical performance limitations of organic fluorophores, including low brightness and low photostability, due to the highly rigid aluminosilicate core surrounding the encapsulated dye, resulting in both increased radiative and decreased non-radiative rates. , In recent work, it was discovered that the four-fold coordinated aluminum in the aluminosilicate core of aC′ dots, along with oxygen naturally dissolved in aqueous solutions, induces optical blinking across a range of encapsulated dyes, likely via photoinduced redox processes, under simple imaging conditions that do not require an additional UV activation laser, cytotoxic thiolated compounds, or oxygen scavengers . This low ON–OFF duty cycle (ratio of “on” time over collection time) optical blinking enables optical super-resolution microscopy (SRM), for example in the form of stochastic optical reconstruction microscopy (STORM), to resolve features below the diffraction limit of visible light. , Furthermore, the enhanced brightness and photostability of aC′ dots relative to their parent dyes make it possible to achieve higher resolutions in STORM reconstructions, based on durable higher photon counts per blink/localization. , Finally, conjugating a sensor dye to the PEG-shell of aC′ dots has subsequently been demonstrated to allow SRM-enhanced ratiometric sensing of metabolic parameters within cells, adding another powerful functionality to these multifunctional optical SRM probes . In order to add highly specific (e.g., intracellular) targeting and immunofluorescent labeling capabilities to this platform, there is a clear need to develop robust protocols allowing efficient conjugation of Abs to the aC′ dot surface.…”