Advances in the development of small photoblinking semiconducting polymer dots (Pdots) have attracted great interest for use in super-resolution microscopy. However, multicolor super-resolution imaging using conventional small photoblinking Pdots remains a challenge due to their limited color choice, broad emission spectrum, and heavy spectrum crosstalk. Here, we introduce two types of small photoblinking Pdots with different colors and relatively narrow emission spectra: blue PFO Pdots and carmine PFTBT5 Pdots for blinking-based statistical nanoscopy. Both of these probes feature ultrahigh single-particle brightness, very strong photostability, superior biocompatibility, and robust fluorescence fluctuation. In addition, these small photoblinking Pdots serve as excellent labels for dual-color super-resolution optical fluctuation imaging (SOFI) of specific subcellular structures, indicating their promise for long-term multicolor SOFI nanoscopy with high spatiotemporal resolution.

Two types of small photoblinking Pdots with high brightness, strong photostability, and favorable biocompatibility, are designed. Super-resolution optical fluctuation imaging is achieved using these Pdots. Imaging of subcellular structures demonstrates that these small photoblinking Pdots are outstanding probes for fast, long-term super-resolution fluorescence imaging.

Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane-substituted AIEgens to generate compact and ultrastable AIE dots via photo-crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation. Due to their small sizes, good stability, and surface functionalization, the cell-surface markers and subcellular structures are specifically labeled by the AIE dot bioconjugates. Remarkably, stimulated emission depletion imaging with AIE dots is achieved for the first time, and the spatial resolution is significantly enhanced to ≈95 nm. This study provides a general approach for small functional molecules for preparing small sized and ultrastable nanodots.

The advance of optical super-resolution fluorescence microscopy has revolutionized our vision of the subcellular world. Further improvement in the spatial resolution is of great significance for structural and functional investigations. The recently developed expansion microscopy (ExM), which achieves sub-diffraction imaging via physical expansion of the sample, provides a great opportunity for further resolution enhancement of existing optical super-resolution techniques. However, although such combination seems apparent, several technical obstacles, especially the dramatic loss of fluorescence signal during ExM sample preparation, have hampered this goal. In this work, aiming at this challenge, we have developed new strategies to retain and increase the fluorescence of the expanded sample. With the new labeling methods, we have successfully made the labeling density of expanded samples sufficing the Nyquist sampling criteria for optical super-resolution imaging, such as stimulated emission depletion microscopy (STED) and super-resolution optical fluctuation imaging (SOFI). The newly developed expansion nanoscopic imaging (ExN) approaches, i.e. ExSTED and ExSOFI, demonstrated up to 4-fold resolution enhancement compared to standard STED and SOFI, providing a simple and effective way to realize high resolution imaging both at the cellular and tissue level.