Multicolor Super-resolution Fluorescence Microscopy with Blue and Carmine Small Photoblinking Polymer Dots

Abstract

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.

Polymer Dots as Effective Phototheranostic Agents

“…Photoblinking is a special kind of optical property, which refers to the sudden change of fluorescence intensity of some nanomaterials (such as quantum dot and polymer dot) from bright to dark or an opposite process. It can not only be used for single molecular identification, but can also be applied in superresolution optical fluctuation imaging . Wu and coworkers reported one type of small photoblinking Pdots with narrow emissive spectra and carmine fluorescence (Fig.…”

Nanoparticle Probes for Super‐Resolution Fluorescence Microscopy

“…Sun and co‐workers,, synthesized Pdots by co‐precipitation of a poly(styrene‐ co ‐maleic anhydride) polymer (PSMA) and a fluorescent polymer (poly[2‐methoxy‐5‐(2‐ethylhexyloxy)‐1,4‐(1‐cyanovinylene‐1,4‐phenylene)] (CN‐PPV) or poly[(9,9‐dioctylfluorenyl‐2,7‐diyl)‐co‐(1,4‐benzo‐{2,1′,3}‐thiadazole)] (PFBT)) and used them for SOFI imaging of subcellular structures. The blinking behavior of these Pdots could easily be fine‐tuned by varying the mass ratio of the two polymers.…”

“…Background‐free 2D and 3D SOFI images of subcellular structures including mitochondria, microtubule filaments, and the nuclear lamina were collected with antibody‐functionalized Pdots (Figure ); essentially no photo‐degradation was found during image acquisition. Pdots with different emission colors, synthesized from the blue‐emitting polymer poly(9,9‐dioctylfluorenyl‐2,7‐diyl) (PFO) and the carmine‐emitting polymer poly(9,9‐dioctylfluorene)‐co‐(4,7‐di‐2‐thienyl‐2,1,3‐benzothiadiazole) (PFTBT5), have been employed for multicolor SOFI imaging of clathrin coated pits and the microtubular network …”

Joint tagging assisted fluctuation nanoscopy enables fast high‐density super‐resolution imaging

“…The spatial resolution improvement comes at the cost of sacrificing the temporal resolution. In recent years, the developments of fluctuation‐based super‐resolution microscopy, such as super‐resolution optical fluctuation imaging (SOFI), super‐resolution radial fluctuation (SRRF), etc., make it easier to obtain a high‐spatial resolution with fewer frame numbers by exploring the inherent blinking nature of fluorophores . SOFI achieves super‐resolution reconstruction through calculating the temporal auto‐cumulants or spatiotemporal cross‐cumulants of the blinking image sequences.…”