Fluorescent Semiconducting Polymer Dots With Modulated Photoblinking For Super-Resolution Microscopy

During the past 50 years,conjugated polymer nanomaterials(CPNs)have witnessed a prodigious growth in the field of organic optoelectronic devices,solar cells,biochemical sensors,cancer therapeutics and biomedical imaging.In comparison with conventional fluorescent probes,such as organic dyes,fluorescent protein and quantum dots(Qdots),conjugated polymer nanoparticles(Pdots)exhibit large absorption crosssection,fast emission rate,tunable emission wavelength and good biocompatibility,which paves the way to the field of life science.Since the advent of super-resolution microscopy in 1990 s,new discoveries have been promoted dramatically in the field of life science.Super-resolution microscopy could bypass the physical barrier of diffraction limit(~250 nm),thus providing an unprecedented view about the subcellular structures and dynamic processes.Stimulated emission depletion microscopy(STED),structured-illumination microscopy(SIM),photoactivated localization microscopy(PALM),super-resolution optical fluctuation imaging(SOFI)and stochastic optical reconstruction microscopy(STORM)have been invented in succession,which greatly enrichs the family of super-resolution microscopy.Among most super-resolution techniques,it is of paramount importance for fluorescent probes to switch reversibly between different states(on/off).Therefore,single-particle brightness,antiphotobleaching ability and fluorescence fluctuation intensity are key parameters for fluorescent probes.It is of great significance to develop highly fluorescent probes with small size,execllent anti-photobleaching ability and tunable photoblinking properties for higher spatial and temporal resolution.We are aimed at the design of conjugated polymer dots with excellent photophysical properties.The main research content in this dissertation are as follows:Single-particle brightness and photostability are prerequisite parameters for fluorescent probes in super-resolution imaging.In this study,we first investigated the underlying physical mechanism of dye-doped Pdots with great photostability.F?rster resonance energy transfer(FRET)is described as a useful strategy to enhance the photostability of fluorescent nanoparticles.Dye molecules were doped into semiconductor polymer dots(Pdots)as FRET acceptors,yielding apparent suppression of the rapid photobleaching in single-particle imaging.For 20-nm diameter particles,the photobleaching percentage decreased from 71.8% to 47.2% after dye doping,while the single-particle brightness remained unchanged.This study indicates that FRET is a facile,yet effective approach to mediate the brightness and photostability of fluorescent nanoparticles.Considering single-particle brightness and photostability,the dye-doped Pdots of ?20 nm diameter are most suitable for long-term single-particle tracking and super-resolution imaging.Based on Monte Carlo simulation,we investigated the photoblinking behavior in polymer nanoparticles and optimized the optical parameters for high-order SOFI nanoscopy.The relationship between the number of chromophores and the amount of active emitting centers was determined in the presence of energy transfer.As with the increasing number of active emitters,the on-time ratio gradually increases from 0.1 to ~0.8.This simulation model is also useful to evaluate the spatial resolution of highorder SOFI nanoscopy at distinct on-time ratio.Best spatial resolution can be obtained by high-order SOFI nanoscopy at the on-time ratio of ~0.4.According to the simulation result,we designed and synthetized a series of conjugated polymers with varying on-chain donor-acceptor moieties by Suzuki crosscoupling reaction,including PF5 BT,PF10BT,PF20 BT and PF50 BT.The photoblinking behaviors are quantitatively compared in terms of autocorrelation function,on-time ratio,powerlaw distribution and dot retention fraction.In this study,photoblinking behaviors are effectively modulated in a nanoscale multichromophoric system consisting of donor-acceptor semiconducting polymers,which is demonstrated in high-order SOFI at a single-particle level.High-performance photoblinking-based SOFI nanoscopy is implemented by using the PF10BT-streptavidin Pdots,resolving microtubular structure in HeLa cell at a spatial resolution of ~95 nm(~4 fold beyond the diffraction limit).Due to the photochromism of diarylethenes,we designed and synthetized a on/off controllable semiconducting polymer DTE-PFBT by covalently coupling this photochromic molecule within the polymer backbone.Under the irradiation of ultraviolet light,DTE-PFBT Pdots turned into a dark state wihout emission as a "closedloop" type(c-DTE-PFBT).Under the irradiation of visible light,DTE-PFBT Pdots recoverd green emission as a "open-loop" type(o-DTE-PFBT).Under alternative laser irradiation at different wavelength,the on/off switching behavior exhibits a fast response rate and resistance to high fatigue,which is evidenced by quantitative spectral analysis.According to the single-particle trajectories,the blinking rate ratio was determined to be >10,indicating that DTE-PFBT Pdots can be applied in STORM.The best spatial resolution of STORM reached at 32.1 nm at single particle level.According to STORM based on DTE-PFBT Pdots,microtulule within BSC-1 cell can be resolved at a spatial resolution of ~40 nm.