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

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.

Ultrasmall, Bright, and Photostable Fluorescent Core-Shell Aluminosilicate Nanoparticles for Live-Cell Optical Super-Resolution Microscopy.

Stochastic optical reconstruction microscopy (STORM) is an optical super-resolution microscopy (SRM) technique that traditionally requires toxic and non-physiological imaging buffers and setups that are not conducive to live-cell studies. It is observed that ultrasmall (<10 nm) fluorescent core-shell aluminosilicate nanoparticles (aC' dots) covalently encapsulating organic fluorophores enable STORM with a single excitation source and in a regular (non-toxic) imaging buffer. It is shown that fourfold coordinated aluminum is responsible for dye blinking, likely via photoinduced redox processes. It is demonstrated that this phenomenon is observed across different dye families leading to probes brighter and more photostable than the parent free dyes. Functionalization of aC' dots with antibodies allows targeted fixed cell STORM imaging. Finally, aC' dots enable live-cell STORM imaging providing quantitative measures of the size of intracellular vesicles and the number of particles per vesicle. The results suggest the emergence of a powerful ultrasmall, bright, and photostable optical SRM particle platform with characteristics relevant to clinical translation for the quantitative assessment of cellular structures and processes from live-cell imaging.

Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments.

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

Dye Encapsulation in Fluorescent Core-Shell Silica Nanoparticles as Probed by Fluorescence Correlation Spectroscopy.

Synthetic advances in the formation of ultrasmall (<10 nm) fluorescent poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles (SNPs), enabling improved particle size and surface chemical property control have led to successful clinical translation of SNPs as diagnostic probes in oncology. Despite the success of such probes, details of the dye incorporation and resulting silica architecture are still poorly understood. Here, we employ afterpulse-corrected fluorescence correlation spectroscopy (FCS) to monitor fast fluorescence fluctuations (lag times <10-5 s) of the negatively charged cyanine dye Cy5 as a probe to study such details for dye encapsulation in 5 nm silica cores of PEGylated core-shell SNPs (C dots). Upon deposition of additional silica shells over the silica core we find that the amplitude of photo-induced cis-trans isomerization decreases, suggesting that the Cy5 dyes are located near or on the surface of the original SNP cores. In combination with time correlated fluorescence decay measurements we deduce radiative and non-radiative rates of the Cy5 dye in these particles. Results demonstrate that FCS is a well-suited tool to investigate aspects of the photophysics of fluorescent nanoparticles, and that conformational changes of cyanine dyes like Cy5 are excellent indicators for the local dye environment within ultrasmall SNPs.

High-Performance Chromatographic Characterization of Surface Chemical Heterogeneities of Fluorescent Organic-Inorganic Hybrid Core-Shell Silica Nanoparticles.

In contrast to small-molar-mass compounds, detailed structural investigations of inorganic core-organic ligand shell hybrid nanoparticles remain challenging. The assessment of batch-reaction-induced heterogeneities of surface chemical properties and their correlation with particle size has been a particularly long-standing issue. Applying a combination of high-performance liquid chromatography (HPLC) and gel permeation chromatography (GPC) to ultra-small (<10 nm diameter) poly(ethylene glycol)-coated (PEGylated) fluorescent core-shell silica nanoparticles, we elucidate here previously unknown surface heterogeneities resulting from varying dye conjugation to nanoparticle silica cores and surfaces. Heterogeneities are predominantly governed by dye charge, as corroborated by molecular dynamics simulations. We demonstrate that this insight enables the development of synthesis protocols to achieve PEGylated and targeting ligand-functionalized PEGylated silica nanoparticles with dramatically improved surface chemical homogeneity, as evidenced by single-peak HPLC chromatograms. Because surface chemical properties are key to all nanoparticle interactions, we expect these methods and fundamental insights to become relevant to a number of systems for applications, including bioimaging and nanomedicine.

Quantitative Comparison of Dye and Ultrasmall Fluorescent Silica Core-Shell Nanoparticle Probes for Optical Super-Resolution Imaging of Model Block Copolymer Thin Film Surfaces.

In recent years, high-resolution optical imaging in the far field has provided opportunities for alternative approaches to nanocharacterization traditionally dominated by electron and scanning probe microscopies. Here, we report the optical super-resolution imaging of model block copolymer (BCP) thin film surface nanostructures through stochastic optical reconstruction microscopy (STORM). We compare a set of surface-functionalized fluorescent core-shell silica nanoparticles encapsulating two different organic dyes, Cy3 and Cy5, with the corresponding free dyes in STORM. Using various click-type chemistries, these probes are covalently attached to the surface of specific blocks of BCP thin films, enabling selective block labeling and optical visualization. We demonstrate that the enhanced brightness of these particle probes offers distinct advantages over conventional dye labeling, outperforming one of the best STORM dyes available (Cy5).

Amorphous Quantum Nanomaterials.

In quantum materials, macroscopic behavior is governed in nontrivial ways by quantum phenomena. This is usually achieved by exquisite control over atomic positions in crystalline solids. Here, it is demonstrated that the use of disordered glassy materials provides unique opportunities to tailor quantum material properties. By borrowing ideas from single-molecule spectroscopy, single delocalized π-electron dye systems are isolated in relatively rigid ultrasmall (<10 nm diameter) amorphous silica nanoparticles. It is demonstrated 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). The 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. The resulting nanoprobes exhibit features proven to enable clinical translation.