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

Abstract

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

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

“…The brightness per ATTO647N in aC′ dots was enhanced on average about 1.5-fold, essentially independent of pH (Figure S2, Supporting Information). This enhancement is a result of the rigidification by the aluminosilicate matrix, as investigated in detail earlier, [19,22d] but was slightly reduced compared to values obtained in previous studies in ultrapure 18.2 MΩ water, [19] likely due to the PBS buffer. When the same measurements were performed for ATTO647N in ultrapure water, the brightness enhancement indeed increased to 1.73 ± 0.11, which is within error from what was previously reported.…”

“…Visibly isolated ROIs were chosen across 3 cells for each pH condition, each cell with 3 ROIs, and the resulting calibrations were generated using averages and standard deviations of the mean pixel values of these ROIs as described later in the Image Processing section. [43] Particle Immobilization for Imaging: Nanosensor particles can be immobilized on glass slides either by inserting biotin into the PEG layer of the nanosensors and binding with streptavidin-coated glass slides as had been used in previous studies, [19,44] or by directly casting nanosensor solution onto glass slides, allowing them to simply adhere to the glass substrate. No appreciable difference in blinking behavior was observed between these methods (data not shown).…”

“…Duty cycles were calculated using previously published methods. [19] Single-particle fluorescence time traces for ≈100 individual particles were generated using a custom Matlab code described previously. [19,44] Duty cycle was calculated using a 100 s sliding window within the 200-400 s time range as described previously.…”

“…STORM: STORM analysis was performed using the ThunderSTORM plugin developed by Ovesný et al [45] As described earlier, [19,44] for optimal localization, maximum likelihood-fitting with a 2D Gaussian PSF was employed. [46] Localizations with lateral uncertainty >5 nm were removed before further analysis.…”

“…[17,18] Furthermore, quantitative analysis of live-cell STORM-imaging data allows estimations of both the sizes of intracellular vesicles containing aC′ dots as well as the number of aC′ dots in each vesicle. [19] Finally, a broad range of organic dyes across different dye families encapsulated in aC′ dots exhibit substantial fluorescence intensity as well as photostability enhancements relative to their parent free dyes in aqueous solutions. [19] All these characteristics make the aC′ dot a desirable candidate for the development of a live-cell ratiometric sensing platform, as it simultaneously overcomes the aforementioned issues of low spatial resolution, signal-to-noise ratio, photostability, as well as biocompatibility.…”