| Cells are the basic structural units that guarantee the normal life activities of organisms.Therefore,a thorough understanding of the various biological events in complete cells is the urgent hope of the scientific community.As an important category of bioassay materials,the invention and creation of fluorescent probes provide a window for people to peek into the inner secrets of cells and make it possible to image,analyze and track living cells,which is equivalent to an "endoscope" for complete cells.With the help of fluorescent microscopy,real-time and in-situ observation of cells can be realized.The small organic molecule fluorescent probe has been widely used due to its unique advantages including simpler staining process,better membrane permeability and more flexible molecular design strategy.Therefore,this paper mainly focuses on the following two aspects:(1)design and synthesis of two kinds of target-switchable fluorescent indicators to detect mitochondrial membrane potential(MMP),(2)design and synthesis two single-molecule/dual-target fluorescence probes for simultaneously and distinctively imaging plasma membrane&lipid droplet and plasma membrane&mitochondria,and providing a powerful imaging tool to further analyze the organelle interaction networks.Mitochondria are important organelles that can carry out metabolism and provide energy for cells.They have negative internal and positive external membrane potentials(MMP)up to-180 mV.Stable MMP maintains normal physiological activities of cells,such as ATP synthesis and cationic passive transport.Abnormal changes in MMP can lead to mitochondrial diseases such as keratitis,Parkinson's disease and cancer.A large number of data showed that the vanished MMP could be a signal of apoptosis.Therefore,detecting the altered state of MMP(normal decrease and disappearance)is very important for biological research and the diagnosis of related diseases.At present,available MMP indicators are either fluorescence intensity indicator or ratiometric dyes,and they indicate the change of MMP by the fluorescence intensity from Rhodamine 123 or fluorescence color ratio from JC-1,respectively.However,their practical applications are tedious and have many limitations.Firstly,the fluorescence intensity is affected by many external factors including the light stability,the laser intensity,and the staining concentration.Secondly,the raitometric indicator displays different fluorescence colors based on molecular aggregation and monomer principle,so the staining concentration is not well regulated and the water solubility is extremely poor.In order to facilitate the detection of changes in MMP status,in this work,two mitochondria&mucleus target-switchable spatially dependent fluorescence indicators(LAD and LAD-1)based on the clear boundary between mitochondria and nucleus were designed and synthesized for realizing the convenient and efficient differentiation of MMP in three states(normal,reduced and disappeared).The experimental results showed that LAD could accumulate in mitochondria selectively when MMP was normal.Also,it would move into the nucleus from mitochondria gradually with the decrease of MMP,and finally it targeted the nucleus exclusively when MMP vanished.So the three status of MMP can be point-to-point indicated just by the spatial locations of the indicator and this indicating is reversible.It is worth noting that LAD could be applied to distinguishing dead from viable cells,which is more convenient than the traditional co-staining method.Finally,we also explored the detection mechanism of this kind of fluorescent indicator,and gave a specific design concept based on the experimental results:the molecule need to have both cationic salts and appropriate DNA binding constants.This laid a foundation for the development of more excellent spatially dependent MMP fluorescent indicator in the future.As mentioned above,the indicator LAD could qualitatively detect MMP,on this basis,we developed a fluorescent indicator to quantitatively indicate MMP.To date,the available MMP parameters are either fluorescent intensity from Rhodamine 123 or fluorescence ratio from JC-1.But as mentioned above,MMP detection effect could be affected by staining concentration,so it is difficult to obtain these physical parameters conveniently and reliably.As is well known,colocalization experiment is a universally accepted tool to judge the localization of fluorescent indicator or probe,and the colocalization coefficient(CLC),as a reliable parameter,can quantitatively indicate the staining specificity of a probe to certain an organelle,which can be facilely obtained by confocal microscopy.So in this work,we use CLC as a new MMP indicator and developed a mitochondria&lysosomes target switchable fluorescent probe(Mito-Lyso)to attain the aim.From the experimental results,Mito-Lyso exclusively stains mitochondria with normal MMP and releases to lysosomes with the decreasing of MMP,and the released Mito-Lyso could reversibly transfer between mitochondria and lysosomes.Thus,CLC of Mito-Lyso and a commercial lysosomal probe(NIR-Lyso)could be as a reliable parameter to establish a negatively correlated relationship with MMP.The results showed that this negative correlation between CLC and MMP was confirmed in a variety of cells and not affected by the staining concentration,which was also the unique advantage of target switchable fluorescent indicator.Furthermore,the design principle of Mito-Lyso has been explained in detail and gave corresponding suggestions on the selection of the second target organelle by comparing with LAD:selecting an organelle close to the mitochondria as the second target group will make CLC more sensitive to the indication of MMP.In cells,various organelles and plasma membrane do not work independently,and they form an organelle interaction network through mutual cooperation to achieve rapid material exchange for performing a variety of biological processes of cell life activities,and the disturbance of interaction network is closely related to the occurrence of many diseases.Therefore,it is necessary to simultaneously image two organelles at least for analyzing the regulatory mechanism of organelle interaction and revealing the role of interaction network in substance transport and organelle homeostasis regulation.At present,the simultaneous imaging and analysis of two organelles in the organelle interaction network only relied on the co-staining experiment.However,the uptake rate and number of these two probes are different,so the distribution of these two probes on different targets is not uniform,which finally makes it impossible to synchronous image and observe the two targets.Thus,in this work,we first proposed and designed two kinds of single-molecule/double-target fluorescent probes for simultaneously and distinctively imaging plasma membrane&lipid droplet and plasma membrane&mitochondria.In the interaction network,the plasma membrane is the protective barrier of cells and plays an important role in maintaining the stability of the intracellular environment.Lipid droplets are dynamic organelles that can store biological lipids,so they are involved in various biological processes such as fatty acid transport and signal molecule regulation on the plasma membrane,and excess cholesterol on the plasma membrane is stored in the lipid droplets,thus there is a close relationship between them.Therefore,herein,based on the similar lipid environment in plasma membrane and lipid drops,using the similar miscibility principle,a long alkyl chain was introduced into the probe as the targeting group,and according to the different water content in them,a kind of single-molecule/double-arget fluorescent probe(PMLD-18)was designed.It broke the traditional way of that one probe can only stain one organelle,and imaging plasma membrane and lipid drops differentially and simultaneously.Experimental results showed that PMLD-18 could simultaneously image the plasma membrane and lipid droplet with green and yellow fluorescence,and in situ spectra showed that the maximum emission peaks between them were 37 nm apart,so the polarity of plasma membrane was higher than that of lipid droplet.In addition,the control molecule M-1 was synthesized to study the targeting mechanism of PMLD-18:long alkyl chain groups are very beneficial for targeting both plasma membrane and lipid droplets.As an energy factory within the cell,mitochondria can provide enough energy for many dynamic activities happened on the plasma membrane,including cell division,partial phagocytosis and proliferation.So there is also close relationship between mitochondria and plasma membrane.To explore the correlation between them,herein,we proposed and designed three single-molecule/double-target fluorescent probes(PMM-1,PMM-2,and PMM-4)based on ICT mechanism for simultaneously imaging plasma membrane and mitochondria for the first time.From the experimental results,all the probes can simultaneously imaging plasma membrane and mitochondria with green and red fluorescence.From the in situ spectrum,the polarity of mitochondria is larger than that of the plasama membrane with a difference of about 45 nm.Moreover,according to the real-color imaging results of PMM-2,the interaction location between the plasma membrane and the mitochondria in the cells was analyzed,and the closest distance between them can be clearly seen as only 400 nm through the super-resolution microscope imaging.Finally,by comparing with the control molecules and PMLD-18,suggestions about designing this type of probe were gaven:(1)positive cation salt with appropriate hydrophobic ion radius is very favorable for targeting mitochondria,(2)the structures containing naphthalene and N,N-dimethyl contribute to targeting the plasma membrane(3)N,N-dimethyl as the electron donor group is beneficial for the solvent chromogenic effect.So that more single-molecule/double-target probes could be designed to provide more imaging tools for analyzing organelle interaction network.In conclusion,in this work,two kinds of target-switchable fluorescent indicators were designed and synthesized for indicating the change of MMP from the different perspectives:easily distinguish the three states of MMP by using spatial position,providing the convenient and reliable parameter CLC as the MMP indicator based on the target switchable advantage.In addition,two kinds of single-molecule/dual-target fluorescence probes were developed for simultaneous and differential imaging of plasma membranes&lipid droplets and plasma membrane&mitochondria.In terms of fundamental research,the design mechanism of the above two types of fluorescent indicators and probes were explored,which laid solid foundation for developing more powerful imaging tools in the future.From the specific applications,these fluorescent indicators and probes have low cytotoxicity and specific targeting capability,so they have great application potential for biological research and the diagnosis of related diseases. |
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