Preparation And Bioimaging Study On Time-resolved Fluorescent Probes Of Organic Small Molecule

Cells in living organisms are composed of highly dynamic biomolecular networks,and organelles are functional regions where eukaryotic cells carry out life activities.Each organelle performs a specific function,and at the same time they are highly coordinated among themselves,regulating multiple biological processes of cellular life activities.In addition,proteins also participate in and regulate basic cellular metabolic activities and signal transmission.Studies have shown that cell homeostasis is a necessary condition for organisms to maintain normal life activities,and its dysfunction is closely related to the occurrence and development of many diseases.However,due to the lack of powerful detection tools to achieve molecular and subcellular level tracing and quantification of organelle interaction networks and metabolites,the study of the patterns and mechanisms of organelle crosstalk and the level of protein metabolism remains a difficult problem.Therefore,aiming at the diversified challenges of intracellular signal detection,this paper developed a series of time-resolved multifunctional small organic molecular fluorescent lifetime probes to deeply study the cellular interaction network and its regulatory mechanisms and accurate quantitative information of metabolites.The results of this study can be summarized in three parts.The details are as follows:In Part Ⅰ:Fluorescence intensity-based probes have been used to study the behavior of intracellular α-chymotrypsin,yet their application for quantitative analysis ofα-chymotrypsin in biological systems is severely limited by their susceptibility to inhomogeneous distribution of probe concentrations.Here,this work presents a non-peptide organic small molecule fluorescent probe(BIQBr)for the first time based on a time-resolved strategy to enable imaging of endogenous α-chymotrypsin in living cells.The probe BIQBr uses an aromatic azonia units as the fluorescent master and connects 4-bromobutylacetyl as the recognition site of α-chymotrypsin.The molecular docking results show that BIQBr has good binding affinity on α-chymotrypsin with a thermodynamic binding energy of-11.74 kcal/mol.The probe shows a significant fluorescence lifetime change when bound to α-chymotrypsin and can be used to visualize α-chymotrypsin in living cells by FLIM.This is the first FLIM-based non-peptide organic small molecule fluorescence lifetime probe for sensing α-chymotrypsin in living cells,and may provide guidelines for the diagnosis and treatment of diseases such as pancreatic cancer.In Part Ⅱ:Organelles are dynamic,yet highly organized to maintain cell homeostasis.However,lack of time-resolved molecular tools for simultaneous dual-signal imaging of two organelles has prevented researchers from elucidating organelle interaction regulatory mechanisms at nanosecond timescale.To date,the interaction regulatory mechanisms between endoplasmic reticulum(ER)and autophagosomes remain unclear.In this work,we propose a strategy to develop dual-fluorescence-lifetime probes located at ER and autophagosomes to explore their interaction regulatory mechanisms.Using the robust probe CF2,we investigated the regulatory mechanisms between ER and autophagosomes,and found that:(i)motile autophagosome in ER tips drive the ER tubule to grow and slide;(ii)ER reticulate tubule forms a three-way junction centered on the autophagosome;(iii)ER autophagy is a kind of cell damage index during druginduced apoptosis.This work not only solves the optical challenge of the existence of signal crosstalk in multi-dye re-stained organelles,but also breaks the limitation that multi-channel switching imaging cannot simultaneously observe organelle interactions,facilitating the study of the regulatory mechanisms of organelle interactions at the cellular level,which is expected to provide insights into the discovery of therapeutic targets for neurodegenerative diseases.In Part Ⅲ:To address the complex testing challenges of being able to quantitatively trace life activities at the molecular level while having subcellular resolution within biological systems,this work presents the first rational design of a multifunctional fluorescent probe with long fluorescence lifetime,resistance to photobleaching and high brightness based on a naphthalimide fluorescent master,enabling time-resolved and super resolution imaging of intracellular substructures.It was found that the fluorescence lifetime of the probe Nap-ER is sensitive to polarity changes and can image lipid droplets and endoplasmic reticulum in a dual-channel,dual-mode manner,making it an excellent tool for direct observation of endoplasmic reticulum and lipid droplet interactions in living cells.In the present work,using Nap-ER,time-resolved and super resolution imaging was performed in cells to investigate the morphological changes in lipid droplet-regulated endoplasmic reticulum stress behavior.It was found that:(1)the number of lipid droplets increased,and their size decreased after tunicamycin-induced endoplasmic reticulum stress;(2)the endoplasmic reticulum tubular structures collapsed around the cell membrane,while the endoplasmic reticulum lamellar structures broadened and extended throughout the cell lumen and became less polar.Thus,after tunicamycin-induced endoplasmic reticulum stress in cells,lipid droplets apparently undergo lipolysis or autophagy and participate in the remodeling of endoplasmic reticulum membrane structural domains in response to environmental changes.The present work reveals a new perspective on the cellular regulation of contacts between these organelles and promises a new understanding in how cells shape the fate of the whole organism.