Construction And Biologicalapplication Of A Novel Fluorescent Lifetime Probe For Cell Microenvironment

Eukaryotic cells contain a variety of organelles,such as mitochondria,lysosomes,vesicles and so on.They complete various life processes through mutual cooperations.They also play an irreplaceable role in material metabolism,cell apoptosis and other aspects,and their internal microenvironment needs to be maintained at a stable level to ensure normal operations.The organelle microenvironment mainly includes p H,polarity,viscosity and other factors.Studies have proved that the pathological factors in many diseases are related to the disorder of microenvironment in organelles.Therefore,it is of great significance to visualize the microenvironment of organelles with appropriate methods.Fluorescence lifetime imaging microscopy(FLIM)technology provides a good way for researchers to explore the microenvironment of organelle in cells.Fluorescence lifetime imaging technology has excellent biological detection advantages,such as immune to changes in excitation luminescence intensity and fluorophore concentration.Therefore,the detection to microenvironment of organelle,such as,lysosomes,vesicles and mitochondria via fluorescence lifetime imaging will have a very broad prospect in the exploration to clinical research.In all,we reasonably designed three fluorescent probes,which are well targeted to organelles,and are respectively used to explore the visual detection of lysosomal p H,vesicle viscosity,and mitochondrial polarity,as well as their application in pathological models.Firstly,the probe PLN has advantages,such as,sensitivity to p H changes and good selectivity.The co-localization experiment shows that PLN can track lysosomes.Most importantly,PLN can quantify the p H value of individual lysosomes through variations in fluorescence lifetime.As the PLN peoforms shorter fluorescence lifetime,it means the p H value of lysosomes is lower.Under the stimulation of different drugs,PLN can detect the p H difference of lysosomes among various cells via FLIM imaging.Most importantly,the probe PLN can evaluate the acidic environment during tumor stratification via FLIM imaging.Secondly,a fluorescent probe IVP-Y is designed to analyze changes in intracellular vesicle viscosity.Based on the molecular structure of D-π-A,the pyridinium salt part acts as the receptor and enhances the water solubility of the probe.The methoxy xanthene derivative plays a key role as an effective electron donor,and the conjugated vinyl bond structure as the molecular rotor will rotate according to the viscosity sensitivity.When IVP-Y is located in a higher viscosity environment,the probe emits fiercer fluorescence enhancement and shows a longer fluorescence lifetime.In addition,IVP-Y can visualize changes in intracellular vesicle viscosity under drug stimulation via FLIM.Finally,the fluorescence lifetime of PEB-apop,a polarity-senseitive probe,decreases significantly with the increasing polarity of the solvent.The probe PEB-apop also targets mitochondria specifically,monitors the changes of mitochondrial polarity in the process of apoptosis induced by mitochondrial oxidative stress,and realizes the quantitative detection of organelle mitochondrial polarity.