Regulation Of Fluorescence Properties Of Polymer Dots And Its Application In Super-resolution Fluorescence Imaging

As human cognition of life deepens,exploring the dynamic process of the smallest cell unit that constitutes an organism is still important.Without the aid of imaging devices,humans cannot resolve tiny cels with naked eyes.In the biomedical research,cel-level studies are normally carried out by means of microscopy,such as the widefield optical microscopy.Due to the diffraction limit of light,this optical microscopy cannot be infinitely resolved.According to the Rayleigh criterion,the resolution of an optical microscopy cannot be smaller than half of the wavelength of visible light,which means that the microscopy cannot resolve objects with a spatial position of 200 nm,even if it is equipped with an objective with large numerical aperture or excited by an ultraviolet with a short wavelength.However,many organelles and molecule structures in cells have a small size of 1~50 nm,which markedly surpass the resolution of conventional optical microscopy.More than a century after the German scientist Ernst Abbe proposed the optical diffraction limit,the super-resolution optical microscope based on different working principles have been developed to shatter this limit.The super resolution imaging can be achieved by four main categories of approaches.The first categories of methods are stimulated emission depletion microscopy(STED)and structured il umination microscopy(SIM),which are based on modulation of point spread function.The second categories are based on photoswitching properties of organic days or fluorescent proteins,including stochastic optical reconstruction microscopy(STORM)and photoactivated localization microscopy(PALM).In addition,super-resolution optical fluctuation imaging(SOFI)based on photoblingking properties of fluorescent probes and expansion microscopy(ExM)based on sample expansion are also widely used in the scientific research.All these methods rely heavily on the optical properties of fluorescent probes,and the chalenge is that the fluorescence brightness and anti-photobleaching properties of commonly used organic dyes and fluorescent proteins need to be improved in the longterm imaging applications.As a new generation fluorescent probes,polymer dots have been widely used in biological imaging,biosensing and optical therapy due to their high fluorescence brightness,high absorption cross section,strong anti-photobleaching properties and biological compatibility.Therefore,it is of great importance to apply polymer dots into the super resolution optical imaging.The results we obtained can be described as below:(1)Cooperative blinking from dye ensemble activated by energy transfer for super-resolution celular imaging.We prepared PFO-Coumarin6 and CNPPV-NIR695 Pdots with pronounced blinking.The optimum doping ratio of dye acceptors were obtained along with avoided aggregation caused quenching.Fluorescent spectra and single-particle fluorescent spectra and single-particle fluorescence blinking show that both these two types of Pdots possess pronounced,high on/off ratio photoblinking property.The fluorescent ‘bright state' of cooperative Pdots comes from dye acceptors and the ‘off state' caused by polymer donors.The photogenerated hole polarons of polymers broken the Fo?rster Resonance Energy Transfer(FRET)process which is sensitively dependent on the distance and spectral overlap between donor and acceptor.The results of single-particle SOFI show that the spatial resolution of fourth-order SOFI images is 68 nm,which is 5.3 times higher than that of conventional wide-field microscopy.We conjugated streptavidin with cooperative blinking Pdots for specific subcellular labeling and obtained 90 nm spatial resolution with forth-order SOFI analysis.(2)We demonstrate dual-color super-resolution optical fluctuation imaging(SOFI)by using two types of narrow-band semiconducting polymer dots(Pdots).We developed two types of narrow emission(FWHM<50 nm)Pdots and named as PF5BODIPY565 and PF8BODIPY720 Pdots.We compared photophysical property of narrow emissive Pdots with commercial dyes,Alexa555 and Alexa647.Narrow emissive Pdots show excelent single-particle brightness and anti-photobleaching property compared with commercial dyes.Both these two types of narrow emissive Pdots are pronounced photoblinking and show a power-law fitting of ‘off state'.In SOFI analysis,the higher order of cross cumulant,the higher resolution of SOFI images.We obtained 57 nm resolution with eighth-order SOFI analysis in single-particle,which is 6-fold enhance compared with conventional wide field microscopy.In the eighthorder SOFI super-resolution images of BS-C-1 microtubules labeled with PF5BODIPY565 Pdots,the spatial resolution of 61-nm microtubules was realized,which was five times higher than the wide field.This resolution is the best one based on Pdots fluorescent probe labeling microtubules.By optimizing the labeling procedure,dual-color eighth-order SOFI imaging of acetylated microtubules and mitochondrial membrane structures in BS-C-1 cels was successfully reoulved based on narrow emissive Pdots,and the spatial resolution of 101 nm was obtained.(3)Pdots was extended to the application of STED nanoscopy.We obtained stimulated emission of polymer in the microcavity whispering galery wals.The stimulated emissive wavelength can be precisely tuned by tuning the geometric size of microcavity.As a gain medium,the polymer showed super photostability.Under the excitation of near-threshold pump intensity,the polymer microcavity laser intensity showed almost no decrease after 20 min.The microcavity lasing of the polymer provides the basis for Pdots application in STED imaging.By the energy transfer with NileRed dye,the STED images of CNPPV-Nilered Pdots labeled BS-C-1 cell microtubule structure were achieved.The spatial resolution was up to 90 nm,which is 2.4 times higher than that in confocal mode.