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

Abstract

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).

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

“…[30][31][32] As shown previously, this brightness enhancement relative to free dye carries over into photon emission enhancements during dye blinking, which is crucial for a potential probe for STORM for which localization precision is proportional to the square root of brightness. [4][5][6] Confocal fluorescence correlation spectroscopy (FCS) measurements on the diffusing PEG-Cy5-aC' dots yielded one-component autocorrelation functions (ACF) suggesting an average particle diameter of 4.6 nm and corroborating the narrow size dispersity reflected in the TEM results ( Figure 1d). We determined an average of 2.5 Cy5 dyes per particle from the combination of FCS derived sample concentration and absorption spectroscopy results.…”

“…While the latter approach removed the requirement for βME in the imaging buffer, a buffer system with an OS system was still employed, as was a two-laser excitation system, all complicating applications in live-cell imaging. [6] Here we describe the surprising discovery that aC' dots, without incorporation of thiol groups and in the complete absence of any particular imaging buffer and under simple illumination with a single light source, exhibit dye blinking enabling STORM based SRM. As we show, the blinking mechanism of aC' dots is very different from that in previous thiolcontaining srC' dots.…”

“…We previously demonstrated that brightness enhancements over free dye indeed directly carry over into photon output enhancements in dye blinking. [ 6,24 ] Taken together, these data point to the emergence of an ultrasmall, bright, and photostable nanoparticle platform for SRM. Building on recent advances, e.g., in bright and photostable nanoprobes including upconversion as well as advanced polymer nanoparticles, ultrasmall aC’ dots constitute an attractive platform for bioimaging and SRM that does not contain heavy metals, exhibits on‐off fluorescence cycling characteristics optimal for STORM, and takes advantage of a wide spectral range that can be tailored by dye choice.…”

“…Optimal STORM probes include those with high brightness, high photostability, and low on-off duty cycle (ratio of "on" over "off" time, vide infra) in cases of high labeling density, as localization precision is proportional to the square root of brightness and low duty cycle stable probes are less likely to have overlapping point spread functions (PSFs). [3][4][5][6] In practice, dye blinking is conventionally achieved by exciting the fluorophores with two different light sources in a complex STORM imaging buffer cocktail, consisting of, e.g., thiol compound beta-mercaptoethanol (βME) and an oxygen scavenging (OS) system. [7] The OS system is typically a combination of glucose, glucose oxidase, and oxygen catalase.…”

“…While the latter approach removed the requirement for βME in the imaging buffer, a buffer system with an OS system was still employed, as was a two‐laser excitation system, all complicating applications in live‐cell imaging. [ 6 ]…”