Study On Single-particle Fluorescence Properties And Regulation Of Polymer Dots

As a new type of biological fluorescence probe, Conjugated polymer quantum dots(Pdots) have been widely applied in fluorescence imaging and long term tracking experiments. Usually, the photophysical property of the Pdots characterized by absorption spectrum and emission spectrum which just show the average effect of the whole Pdots suspension. However, the distribution of the particles were not an average effects when the Pdots were applied to the single-particle and many types of super-resolution imaging experiments. So, that’s a very important work to study the single-particle fluorescent photophysical property of Pdots.In this article, we prepared Pdots with a gradient of particle size by optimizing the conventional “nano-reprecipitation”. We named the Pdots as PFBT12, PFBT21, PFBT37 and PFBT58 according the average distribution of Pdots size. Based on these Pdots, we established the relationship between the per-particle brightness and diameter of the Pdots by using a custom built Total Internal Reflection Fluorescence Microscopy(TIRF-M) system. All of the four Pdots with different size show a simaliar quantum yield(~30%) estimated by the emission spectrum. As the single-particle brightness increases nonlinearly with the diameter. The Pdots were divided into three groups, all of the single-particles fluorescence imaging datas were collected under the same conditions. After the three-group comparisons, the single-particle imaging brightness of the four types of Pdots were back-calculated by the respective intensity ratio and scaled up on the basis of the brightness intensity of PFBT12. The results indicate that there is a quadratic dependence of the single-particle brightness on particle diameter. The relationship curves can serve as a calibration curve for determining the diameter of an unknown particle while under a same experiment condition.We extracted the particle size according to the calibration curve with the given experiment conditons. Then we analyzed the photobleaching behaviors of different types of Pdots. The photobleaching processes can be mainly classified into three types. The results of photobleaching-rate constant and the whole photons number show that, there is not an obvious dependence of the photobleaching-rate constant on the particle size for small size Pdots(<30nm) while there is a fast decay component of the fluorescence intensity for large Pdots(>30nm). The whole photons per-particle emitted show an quadratic relationship on the particle size for small Pdots(<30nm) while the whole photons of the larger Pdots deviated from the quadratic relationship. These results provides a useful guidance for selecting appropriate Pdots(~30 nm) for single-particle imaging and tracking applications.The Pdots show a photobleaching behavior while performed on the single-particle tracking experiments. Some of the Pdots hold the fast decay component. To improve the photostability of the Pdots, we prepared an Oxygen Scavenging System based on enzymatic reaction. The Glucose can reacted with the oxides which usually serve as the fluorescence quanchers fast with the exist of the Glucose Oxidase(GOX). The experiments results indicated that the Oxygen Scavenging System improved the photostability of the Pdots. The improved photostability of the Pdots can make the Pdots show more excellent applications on single-particle imaging and tracking experiments.