Semiconducting Polymer Dots with Modulated Photoblinking for High‐Order Super‐Resolution Optical Fluctuation Imaging

Abstract

Super‐resolution optical fluctuation imaging (SOFI) uses photoblinking to achieve fast, background free, 3D fluorescence nanoscopy. The on/off photoblinking characteristics are key factors for realizing high‐order SOFI. However, it remains a great challenge to tune the photoblinking of a fluorescent probe because most of the probes are typical single fluorophore systems with stochastic blinking nature. In this study, effective photoblinking modulation is demonstrated in a nanoscale multichromophoric system consisting of donor–acceptor semiconducting polymers. Numerical simulation is performed to quantitatively evaluate the impact of the number of acceptors on the photoblinking of the nanoparticle probe as well as the spatial resolution of the resulting SOFI nanoscopy. In tandem with simulation, a series of polymer dots (Pdots) are designed with varying donor–acceptor moieties that give rise to the modulated photoblinking behaviors, as evidenced by single‐particle fluorescence trajectories. The photoblinking behaviors are quantitatively compared in terms of autocorrelation function, dot retention fraction, and on/off time ratio. High‐performance photoblinking‐based nanoscopy is implemented by using the optimized Pdots, resolving subcellular structure at a spatial resolution of ≈95 nm. This study provides a useful approach to engineer the photoblinking properties of fluorescent nanoparticles for super‐resolution imaging applications.

Conjugated Polymers: Optical Toolbox for Bioimaging and Cancer Therapy

“…Furthermore, effective photoblinking modulation of Pdots was achieved by adjusting the D-A ratio in the polymer. [92] It finally provides a spatial resolution of ≈95 nm at the subcellular level and a fourfold enhancement nearly in resolution compared to that of a diffraction-limited image.…”

Optimal correlation order in superresolution optical fluctuation microscopy

“…Besides, for weak sources and detectors with finite efficiency, the absolute values of cumulants rapidly decrease with the growth of their order n. Thus, the resolution enhancement can be also spoiled for a number of reasons mentioned in the Introduction, such as detectors imperfections and additional noise, emitters bleaching or other degradation of sources, image artefacts, etc. [6,10,16,17,[25][26][27][28][29][30][31][32].…”

“…Initially, SOFI was demonstrated with blinking quantum dots (QDs) [1]. Soon, it was applied using organic dyes [15], fluorescent proteins [7,10,16], and semiconducting polymer dots [3,17,18]. Later, SOFI was shown to work by speckled illumination with fluorophores without natural blinking [19,20].…”

“…So, quite lot of research effort was spent in attempts to retrieve potentially infinite resolution enhancement predicted for SOFI by overcoming possible practical limitations in measuring high-order correlations, such as additional noise by imperfections of set-ups, finite size of detector pixels, emitters degradation, acquired image artefacts, etc. [6,10,16,17,[25][26][27][28][29][30][31][32]. Some effort was also spent on enhancing temporal resolution, i.e., on reducing the number of raw images [10,27,33,34].…”

Core size does not affect blinking behavior of dye-doped Ag@SiO2 core–shell nanoparticles for super-resolution microscopy

“…6,[10][11][12][13][14] In recent years many blinking nanostructures made from varying materials such as carbon dots, polymer dots, organic dyes, photoswitchable uorescent proteins, and uorescent nanoparticles have been studied. [15][16][17] The duty cycle and intensity during the "on" states are critical parameters in super resolution microscopy. 13,16 While the mechanism of blinking is still unknown, a number of hypotheses have been presented.…”