| Fluorescent labeling is one of the most effective and efficient biomarker methods,which originated in the 1940s and was first used in fluorescently labeled antibodies to detect the corresponding antigens.With the development of modern medicine,molecular biology and the application of advanced fluorescence detection technology and instruments,fluorescent labeling as a non-radioactive marking technology has been widely concerned and has developed rapidly due to its simple operation,high stability,high sensitivity and high selectivity etc.With the development of biological microscopic imaging technology,two-photon fluorescence(TPM)has become a novel generation of microscopic imaging tools for living cells and tissues.TPM has revolutionized the real-time in vivo visualization of living organs and tissues,particularly that of thick and highly scattering specimens.Compared with single photon laser confocal microscopy,two-photon microscopy has the characteristics of near-infrared light excitation,avoiding fluorescence bleaching and phototoxicity,high resolution,reducing the absorption of excitation light and reducing tissue spontaneous fluorescence interference.However,the advantages of TPM can be severely compromised if the two-photon excited fluorescence action cross-sections(δ×Ω)of the extrinsic fluorophores used as biosensors are smaller than or similar to the endogenous fluorophores.Theoretically,as the single-photon excitation and two-photon excitation obey different selection rules,one cannot expect conventional one-photon dyes to necessarily have large two-photon absorption cross-sections(δ).At present,one photon fluorescent biomaterials which were used in laser confocal microscopy almost do not possess large δ×Ω Therefore,the development of excellent two-photon fluorescent biomaterials is becoming a research hot-topic.In this paper,we are focusing on the integration of analytical chemistry,organic chemistry,biology and materials chemistry to conquer the above bottle-necks in the development of two-photon fluorescent biomaterials for bioimaging based on benzothiazole or benzimidazole.Around this purpose,this paper mainly carried out the following work:(1)A series of novel membrane permeable two-photon fluorescent dyes were designed and synthesized,and its photophysical properties,cell imaging experiments and membrane permeable of the dyes were carried out.Compared with the classic coumarin,fluorescein and rhodamine dyes,BTVPA,BIVPA,BTPA,BTVCZ,BIVCZ and BVCZ have significant advantages as follows:the two-photon absorption cross-section is much larger than the above three classic dyes and the membrane permeability of the dyes were still retained.BTVPA dye also possesses multiple efficient reaction sites that could be modified to two-photon probes by linking the targeting groups.On the basis of the dyes,we further constructed BTVPA-nucleic acid based on BTVPA dye platform.Two-photon fluorescence imaging showed that BTVPA-nucleic acid could high fidelity detect the nuclei in living cells.(2)Six RNA probes DHTI,DMTI,DMI,IMT-E,IMT-M and IMI were designed and synthesized.In vitro RNA fluorescence titration experiments have shown that the probes have a sensitive optical switch performance,also showed that the high selectivity for RNA.Clear imaging pictures of RNA in nucleolus and cytoplasmic were obtained under single and two-photon microscopy,which demonstrated the selectivity and excellent membrane permeability of the probes.RNA digestion experiments further demonstrated that these six probes can preferentially select endogenous RNA,and there could bind to endogenous DNA when RNA is hydrolyzed.The morphology,size,and number of nucleolus are closely related to the malignant transformation of cells.The morphology of cytoplasm and nuclei and nucleoli information can be obtained at the same time when co-stain with DNA probe.So,though the co-stain fluorescence image picture of using the probe DHTI or DMTI which is red emission and Hoechst 33342 in living or fixed HeLa cells under wide field,confocal or two-photon fluorescence microscope,we could not only observe the quantity,the size and the form of nucleolus,but also the morphology of nucleus and cytoplasm were obtained.It may be provide researchers with an important reference for the diagnosis of cancer.(3)Since the dynamic changes in mitochondria are mostly caused by DNA involvement,mitochondrial DNA probes will provide important support for scientific research.Two red emission,two-photon mitochondrial DNA probe DMT-M and DMT-E were successfully designed and synthesized.In vitro DNA fluorescence titration experiments have shown that the probes possess a sensitive optical switch performance.Clear imaging pictures of DNA in mitochondria which present the filaments shape were obtained under single and two-photon microscopy,which demonstrated the selectivity and excellent membrane permeability of the probes.DNA digestion experiments further demonstrated that these six probes can preferentially select endogenous DNA,and its fluorescence intensity is decreased obviously when DNA is hydrolyzed.Double assay of DMT-M or DMT-E and SYTOX Blue nucleic acid stain(S-11348)showed that the probe possesses excellent membrane permeability to living cells.Moreover,DMT-M and DMT-E exhibit good photostability when continuously illuminated by constant femtosecond pulses.(4)According to the endoplasmic reticulum is also a membrane structure with phospholipid molecular layer,on the basis of the membrane probe,red emission,two-photon endoplasmic reticulum probe MDT were designed the synthesized using the characteristics of membrane probe.Clear imaging pictures of endoplasmic reticulum which present the reticular shape were obtained under single and two-photon microscopy,which demonstrated the selectivity and excellent membrane permeability of the probes.MTT experiment demonstrates low toxicity and biological compatibility of the probe.Due to the mitochondria of immobilized cells do not possess membrane potential,the interference of intracellular mitochondria of MDT was excluded by single and double photon imaging experiments of the fixed cells.Our systematic study will provide ideas to design membrane-targeted endoplasmic reticulum probe.In this paper,a series of two-photon dye platform,RNA probe and mitochondrial DNA probe were designed and synthesized,and the two-photon properties of these fluorescent materials were studied detailedly.The two-photon absorption cross sections of these dye platforms are much larger than classical rhodamine,coumarin and fluorescein dyes,and retain the membrane permeability of classical dyes.The mechanism of recognition of these six RNA probes and RNA was discussed,and though the co-stain fluorescence image picture of using the probe DHTI or DMTI which is red emission and Hoechst 33342 in living or fixed HeLa cells under wide field,confocal or two-photon fluorescence microscope,we could not only observe the quantity,the size and the form of nucleolus,but also the morphology of nucleus and cytoplasm were obtained.It may be provide researchers with an important reference for the diagnosis of cancer.In addition,mitochondrial DNA probes DMT-M and DMT-E were designed and synthesized by adjusting the ability of cationic salt to bind to DNA,and the morphological changes of mitochondria could be tracked for a long time.This paper provides an important basis for the design and synthesis of two-photon fluorescent biomaterials,which will promote the application of two-photon microscopy in biomimetic imaging. |
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