Development Of New Organic Fluorescent Probes For Fluorescence Imaging Of Cellular Lipid Droplets

Lipid droplet is an important organelle,which is composed of neutral lipid nucleus and phospholipid monolayer.It exists in most eukaryotic cells.Lipid droplets vary in size from cell to cell,with large lipid droplets reaching 200?m in size and small new lipid droplets only 30-60 nm in size.Lipid droplets participate in many physiological processes in cells,and the dysfunction of lipid droplets is related to many diseases.Therefore,the study of lipid droplets is imperative.Fluorescence imaging is one of the most powerful tools for observing lipid droplets and studying their versatile functions.Fluorescence imaging technology includes two important elements,one is fluorescence probe,and the other is fluorescence microscope.The resolution limit of traditional fluorescence microscope is about 200 nm due to the optical diffraction limit.However,various super-resolution fluorescence microscopes can break the optical diffraction limit and achieve resolution below 200 nm.The 2014 Nobel Prize in Chemistry was awarded to three physicists for their ingenious ways of circumventing optical diffraction limits by using fluorescent probes to achieve nanoscale resolution with light microscopes.In terms of physics itself,there is no fundamental limitation,the core of their technology is to adjust the luminescence characteristics of the fluorescent probe,to achieve the conversion between light and dark.Ultimately,what had been a purely physical problem became a chemical one:the need to synthesize such fluorescent probes with fluorescent switching properties.This paper aims to regulate the luminescence characteristics of organic small molecule fluorescent probes through reasonable molecular design,develop lipid droplet fluorescent probes that can meet the needs of different fluorescence microscopy,and utilize advanced Stimulated Emission Depletion(STED)super-resolution imaging technology.We can clearly observe the spatial distribution of lipid droplets in cells,accurately track the dynamic process of lipid droplets in cells at the nanometer scale,understand the activity law of lipid droplets in cells,and provide a powerful tool for the study of cell biology of lipid droplets.The details are as follows:a)In order to solve the problem of insufficient staining selectivity of existing commercial lipid droplet fluorescent probe,we developed a new lipid droplet fluorescent probe Ph-Red based on single benzene ring framework.The probe has a simple molecular structure and is easy to synthesize.More importantly,it is based on the small conjugated skeleton of a single benzene ring,which can exhibit efficient red emission.Red emission has a great advantage for fluorescent probes because it can effectively reduce the interference of blue background signal in the cell.The fluorescent probe Ph-Red has high lipid droplet staining selectivity and good biocompatibility,and can be used for repeatable fluorescence imaging.Due to the advantages mentioned above,Ph-Red was used for lipid droplet fluorescence imaging.3D confocal imaging of fixed cells and multicolor confocal imaging of living cells were successfully realized,highlighting the utility of Ph-Red in lipid droplet fluorescence imaging.In addition,we reveal the key factors for efficient staining of cell lipid droplets with fluorescent probes:appropriate hydrophilicity and molecular structure size.These results have important implications for the development of new lipid droplet fluorescent probes.b)In order to solve the problem of inadequate photostability of fluorescent probe for super-resolution imaging of lipid droplet,we developed a fluorescent probe for super-resolution imaging of lipid droplet based on stilbene derivative Lipi-DSB.By introducing an electron-accepting cyanide group and an electron-donating amino group into the two ends and the middle part of the stilbene framework,the obtained Lipi-DSB with the donor-acceptor structure has a high fluorescence brightness and guarantees a large Stokes shift.The introduction of cyanide group can effectively reduce the LUMO level and significantly improve the photostability of Lipi-DSB,while the introduction of amino group can adjust the selectivity of Lipi-DSB.In addition,the probe has low STED laser saturation intensity and good biocompatibility.Based on the above excellent properties,we applied the fluorescent probe Lipi-DSB to STED super-resolution fluorescence imaging of lipid droplets,and obtained the state-of-the-art super-resolution imaging results at that time:1)The highest super-resolution imaging resolution of lipid droplets was 58 nm;2)Time-lapse STED super-resolution imaging of lipid droplets in living cells with the maximum number of images(1000 images);3)The fusion process of nascent lipid droplets was observed for the first time.4)The highest quality 3D STED super-resolution imaging of lipid droplets.Thus,we observed the spatial distribution of intracellular lipid droplets with unprecedented nanoscale resolution and traced the intracellular dynamics of lipid droplets,highlighting the usefulness of the fluorescent probe Lipi-DSB in lipid droplets STED super-resolution fluorescence imaging.c)In order to further improve the super-resolution imaging result of lipid droplet,we developed a fluorescent probe C₂-t Bu QA based on quinacridone derivative with a long fluorescence life(about 20 ns)for biological application of dynamic tracking of intracellular lipid droplet under starvation condition.Long fluorescence lifetime is crucial to improve the resolution of STED super-resolution imaging:on the one hand,it can effectively reduce the saturation intensity of the fluorescent probe,and on the other hand,it allows the use of time-gated detection to further improve the resolution of the imaging.Therefore,we selected on the design of the fluorescent probe has a long life of fluorescent sinensis acridone skeleton,through in the molecular skeleton tert-butyl and alkyl chain,introduced respectively,in promoting fluorescence probe solubility and adjust the hydrophobic at the same time,also can replace active reaction sites on the H atoms enhance the photostability of fluorescent probe.The C₂-t Bu QA fluorescent probe also displayed high fluorescence brightness,good biocompatibility and high lipid droplet staining selectivity.In view of the above advantages,C₂-t Bu QA was used for STED super-resolution imaging of lipid droplets,and the maximum resolution of the super-resolution imaging of lipid droplets was significantly increased from 58 nm to 37 nm,far below the optical diffraction limit.We also accurately tracked the dynamics of lipid droplets at the nanoscale with the highest dynamic imaging resolution(38 nm)over a long period of time(500 STED super-resolution images were taken).During this period,we also detected the first nanoscale lipid droplet fission process.More importantly,we also realized biological applications of fluorescent probe C₂-t Bu QA:Using 4D confocal imaging and dual-color STED super-resolution imaging,we successfully mastered the changes of lipid droplet number and size in He La cells under different starvation conditions,providing a powerful tool for the study of lipid droplet cell biology and promote the development of lipid droplet cell biology.To sum up,this paper developed three new organic small molecules lipid droplets fluorescent probes,solves the existing lipid droplets fluorescent probes staining selectivity and lipid droplets super resolution imaging fluorescent probe serious lack of problem,achieved the highest lipid droplets super resolution imaging resolution and long-term dynamic tracking accuracy of lipid droplets,and successfully realized the biological application of fluorescent probe.The development of these three fluorescent probes for lipid droplet provides a powerful tool for the study of lipid droplet cell biology and promotes the development of lipid droplet cell biology.