Application Of Twisted Intramolecular Charge Transfer Based Probe In Fluorescence Lifetime Imaging

Fluorescence imaging provides a powerful tool for studying the dynamics of intracellular molecules and organelles,especially the development of super-resolution fluorescence technology has greatly improved the temporal and spatial resolution of fluorescence imaging.However,imaging based on fluorescence intensity is also confronted with difficult difficulties.For example,fluorescence intensity depends on probe concentration,and the uptake of probe by cells,distribution of probe in cells,and photobleaching of probe molecules will affect probe concentration,making fluorescence intensity unable to accurately measure real-time fluctuations of intracellular biomolecules.Moreover,most fluorescent probes based on chemical reactions are irreversible,and their fluorescence intensity can only represent the accumulation of the molecules to be measured over a period of time,and cannot measure the real-time fluctuations of biomolecules.In contrast,fluorescence lifetime-based imaging is independent of the concentration of the probe and only closely related to its microenvironment,thus enabling dynamic tracking of subtle changes in the level of the biomolecule under test.In this paper,a series of TICT probes were designed and synthesized based on the application of fluorescence lifetime imaging in cells,which realized the real-time tracking of endoplasmic reticulum,lipid droplets,mitochondria and other organelles and their internal biomolecular dynamics.The main research contents are as follows:1.Monitoring the dynamic of fatty acids in lipid droplets by small molecule probe via FLIM imagingFatty acids dynamic in lipid droplets affects many biological processes such as energy storage,signal transduction,and immune regulation.Understanding metabolism-regulatory organelle behaviors are important in the studies of cellular physiology and pathology,but remains a challenge to fluorescent imaging.The key difficulty is that fluorescent intensity signals are not suitable for monitoring the subtle and real-time fluctuations of metabolic molecule level,due to the uncontrollable cellular uptake and photobleaching of probes.Here,we report a quantitative fluorescence lifetime probe that can track the lipolysis of triglycerides into fatty acids in LDs.The N atom on the nitro group in DPN can form hydrogen bonds with fatty acids.With the increase of fatty acids,the fluorescence lifetime of DPN decreased from1.93 ns to1.21 ns with the excitation at 405 nm.Using DPN to monitor fatty acids dynamic in LDs,we found an obvious increase in fatty acids in cellular apoptosis.Especially,we have proved that the inhibition of fatty acids could alleviate the apoptosis,which provides a useful method to study the dynamic of fatty acids.2.Monitoring lipid droplets viscosity changes during various cell death processes by a near infrared probe via dual modal imagingLDs are complex,dynamical and multifunctional organelles for storing neutral lipids in live cells.Abnormal lipid storage may cause abnormal viscosity in LDs,closely related to various metabolic diseases,such as fatty liver,diabetes and so on.Therefore,real-time and in situ monitoring of LDs viscosity is crucial for studying the mechanisms of some physiological processes and for early detection of related diseases.Herein,we report a viscosity-sensitive NIR fluorescent probe ZYP that is specifically enriched in LDs and exhibits high sensitivity to viscosity.We clearly monitored changes in lipid droplet viscosity during various physiological processes via fluorescence imaging and FLIM imaging,and successfully differentiated normal cells from cancer cells.Interestingly,in cellular experiments,we found that LDs viscosity increased during apoptosis,ferroptosis and autophagy.Overall,the designed ZYP can not only precisely monitor the viscosity changes in LDs during cell death,but also provide guidance for further studies on the important roles of lipid droplets in related physiological processes.3.Revealing the endoplasmic reticulum,lipid droplet,extracellular vesicle dynamics by single molecule probe via FLIM imagingLipids are the basis of cell activities,including neutral lipids and phospholipids.Neutral lipids are stored in LDs,while phospholipids are the main components of cell membranes,organelle membranes,and intracellular and extracellular vesicles.However,the study of lipid metabolism and the differentiation of different phospholipid structures still face great challenges.In this chapter,we designed and synthesized a hydrophobic near-infrared fluorescent probe ZYO,which can locate LDs,ER and extracellular vesicles,and distinguish them by FLIM imaging.ZYO can not only produce hydrogen bonds with fatty acids in LDs to reduce the fluorescence lifetime,but also increase the fluorescence lifetime with the in high order of the phospholipid structure.In the process of apoptosis,the relationship among the production of extracellular microvesicles,the number of LDs and the fatty acids in the lipid droplets was revealed.It was discovered that cells produce vesicles with obvious differences in order during apoptosis,while neutral lipids are secreted to the cell membrane during ferroptosis.ZYO provides an effective tool for further study of lipid metabolism and the biological role of lipid structure.4.Monitoring viscosity changes in mitochondria and in mice liver by BTX via four-mode imagingViscosity in biological systems is a critical factor for various physiological process,including signal transduction and metabolisms of substance and energy.Abnormal viscosity has been proven as a key feature of many diseases,thereby real-time monitoring of viscosities in cells and in vivo is important to understand the mechanism of some diseases and take relevant measures to treat them.Up to date,it is still challenging to monitor viscosity cross-platform from organelles to cells to animals with a single probe.Here,we report a benzothiazoliumxanthene probe with rotatable bonds that switch on the optical signals in high viscosity environment.The enhancements of absorption,fluorescence intensity and lifetime signals allow to dynamically monitoring the viscosity change in mitochondria and cells,while near infrared absorption and emission facilitate imaging the viscosity with both fluorescence and photoacoustic imaging in animals.The cross-platform strategy is capable of monitoring the microenvironment with multifunctional imaging across various levels.