| Lipid droplets are the main organelles in the balance of lipid and energy in cells,and their dysfunction will lead to various diseases.The development of optical microscopy has promoted the study of the physiological function of lipid droplets.However,the specific mechanism of lipid droplets in biological systems is still unclear.In order to further study lipid droplets,advanced microscopic imaging techniques and excellent fluorescent probes of lipid droplets are indispensable.However,traditional fluorescent probes of lipid droplets have some obvious shortcomings,such as low staining signal-to-noise ratio,weak photostability,and the difficulty of tracking lipid droplets in biological bodies due to aggregation induced quenching(ACQ).These shortcomings make the traditional fluorescent probes unable to meet the demanding requirements of advanced microscopic imaging technology.In order to solve this problem,it is necessary to synthesize different types of fluorescent probes suitable for advanced microscopic imaging techniques,such as probes suitable for STED and SIM super resolution imaging.The aim of this paper is to develop lipid droplets fluorescent probes that can overcome the shortcomings of traditional fluorescent probes by using reasonable molecular design,observe more dynamic processes of lipid droplets by using advanced fluorescence imaging technology,so as to provide imaging basis for further study of different physiological functions of lipid droplets.The specific research content of this paper is as follows:(1)In order to solve the problem of low staining signal-to-noise ratio of traditional fluorescent probes,a new lipid droplet fluorescent probe Lipi-Cz-2 based on the sulfuryl bridged stilbene skeleton was developed.By comparing the photophysical properties of Lipi-Cz-2 with that of its isomer Lipi-Cz-1,it can be inferred that LipiCz-2 has TICT property,and Lipi-Cz-1 has ICT property.Then,by comparing the difference between Lipi-Cz-2 and Lipi-Cz-1 in lipid droplets staining signal-to-noise ratio,we concluded that the enhancement of lipid droplets staining signal-to-noise ratio was mainly caused by the formation of TICT state.Lipi-Cz-2 also showed good photostability and biocompatibility,which is very favorable for STED super resolution imaging.By applying Lipi-Cz-2 to STED super resolution imaging,the resolution of lipi-Cz-2 can be improved to 65 nm.In addition,we successfully observed the distribution of intracellular lipid droplets on the nanoscale through 3D STED super resolution imaging of lipid droplets.This work promoted the formation of TICT state through rational molecular design,and provided a new idea for improving the staining signal-to-noise ratio of lipid droplet fluorescent probe.(2)In order to solve the problem of inadequate photostability of traditional fluorescent probes,a new lipid droplet fluorescent probe Lipi-Deep Red based on the sulfuryl bridged stilbene skeleton was developed.The probe significantly improves photostability by introducing p-cyanophenyl groups to protect the active site of sulfuryl bridged stilbene and using diphenylamine donor instead of diethylamine donor.Using Lipi-Deep Red for SIM super resolution imaging,the resolution was successfully improved to 98 nm,much higher than that of wide field microscopy(358 nm).More importantly,the excellent photostability of Lipi-Deep Red enable the fluorescent probe to observe the fusion process of small lipid droplets at the nanoscale in the SIM super resolution imaging.Based on time-lapse SIM super-resolution imaging,the fast fusion processes of LDs(only costing 0.25 s)have been thus precisely visualized.Finally,two contact modes between lipid droplets and mitochondria were observed using two-color SIM super resolution imaging,which provides imaging support for the two contact modes between lipid droplets and mitochondria reported in recent literature.(3)In order to better track lipid droplets in vivo,researchers usually use the nano precipitation method to prepare lipid droplet fluorescent probes into nanocrystals.Unfortunately,ACQ usually occurs during the preparation of nanocrystals by traditional lipid droplet fluorescent probes,resulting in a significant reduction in the fluorescence intensity of nanocrystals,which is very adverse to the tracking of lipid droplets in vivo.To solve the aggregation-induced quenching problem of lipid droplet fluorescent probes,molecule 4 with a highly efficient deep red/NIR emissive organic solid based on stilbene skeleton was developed.By introducing electron-accepting phenylacetonitrile moieties and electron-donating diphenylamino groups into the stilbene skeleton,fluorescent molecule with highly twisted conformation was obtained.Then,by simply altering the terminal alkyl chains,the rotation of phenylacetonitrile group was triggered,which further led to the rotation of diphenylamino group.These rotation changes significantly change the molecular conformation of fluorescent molecule,and thus affected the excited state properties and emission characteristics of fluorescent molecule.It is noteworthy that the fluorescence quantum efficiency of molecule 4represent the highest values among organic solids with similar deep red/NIR emission wavelengths.In view of the excellent solid-state luminescence properties of molecule4,the nanocrystals were prepared using nano-precipitation method,and further verified the low cytotoxicity and high selectivity of lipid droplets staining of the nanocrystals of molecule 4.In conclusion,three new lipid droplets fluorescent probes were developed based on the stilbene skeleton in this paper,which provided a solution to solve the problems of low staining signal-to-noise ratio,inadequate photostability and the difficulty of tracking lipid droplets in biological bodies due to aggregation induced quenching(ACQ)of traditional lipid droplets fluorescent probes.It not only realized the observation of lipid droplet dynamics and the interaction between lipid droplet and mitochondria at the nano scale,but also provided a possible probe for tracing lipid droplets in vivo.The development of three new lipid droplet fluorescent probes provides a new design strategy for the development of new lipid droplets probes,which is conducive to deeper research on lipid droplet biology. |
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