| Recently, a comparison study showed that two-photon fluorescence microscopy (TPM) is a better technique than confocal microscopy in imaging living specimen. TPM produces low photodamage, reduced photobleaching, high detection sensitivity, and no image distortion. In addition, it not only has an intrinsically high axial resolution without the need of a confocal pinhole in the detection path, will but also reduce cell damage significantly due to both the use of longer wavelength excitation light, which avoids damaging ultraviolet (UV) or blue excitation light, and the reduction of out-of-focus irradiation. Consequently, TPM is appropriate for the repetitive imaging of living cells without seriously damaging cellular vitality. To meet the current situation, development of various two-photon excitied fluorescence (TPEF) probes to different targets in living cells have been noticed extensively and intensively. Currently, the status that TPM lacks of excellent two-photon fluorescence probes in the three-dimensional imaging detection of cells and tissues is an urgent prolem to solve. This thesis mainly discusses the three different kinds of fluorescent probes.First, this thesis introduces the membrane probe. Biological membranes are self-assembled bilayers of biomolecules such as lipids, proteins, and carbohydrates. Among such complicated structures, lipids are the major constituents of the membrane structural backbone, and the lipid composition greatly affects membrane functions and properties. According to recent biological membrance theory, there are ordered (Lo) domains enriched in saturated lipids (mainly sphingolipids) and sterols (mainly cholesterol) and disordered (Ld) domains in unsaturated lipids and cholesterol. Moreover, the presence of Lo domains (or rafts) in cell plasma membranes was hypothesized from the discovery of detergent-insoluble membrane fractions enriched with sphingolipids, saturated phospholipids, and cholesterol. This hypothesis stimulated intensive research and debates, since their evidence was mainly provided from indirect techniques. It cannot realize direct visualization Lo domains. At the same time, some researchers think that these domains take part in membrane-associated events such as signal transduction, cell adhesion, signaling, cell trafficking and lipid/protein sorting. Up to now, many researchers use artificial membrane technology to simulate the Lo domains of the cell membrane and carry out various research work. But their direct visualization Lo domains remains a challenge, this state stimulates intensively research on Lo domains of biological membrane and results in ardent debates. Therefore, to understand its role in biology, it is crucial to visualize such domains in living cell membrane. At present, no biochemical techniques are available for such study owing to the difficulty in working with Lo phase in living cell membrane and tissues.Herein, to achieve selectively two-photon imaging Lo domain in living cell membrane, In the second chapter, we design and synthesize a fluorescent probe2,7-bis(1-iodododecane-4-vinylpyridium iodine)-N-ethylcarbazole (HVC-12), and selectively image the Lo phase in plasma membranes of living cell and tissue based aggregation-induced emission (AIE) mechanism. Due to AIE feature of HVC-12, its nanoaggregates accumulate and light up Lo phase of the cell membranes. This thesis firstly found that the Lo phase exhibit uncontinuous distribution (1-1.5μm interval) in the cell membranes. Moreover, compare to commercially available membrane tracers (e.g. DiD), this probe possesses high specificity to live-cell membrane, large stokes shift and two-photon excitation fluorescence action absorption cross-section (δΦ=189GM in EtOH), superior photostability, and well-suited imaging and tracing dynamic change of cell membrane in a long period of time. Many attractive photophysical qualities of HVC-12make it a new powerful tool to study biological membrane.Second, the nucleic acids are important biological macromolecules in cells, and the quantitative analysis and specific recognition of nucleic acids have very important significance to the development of genomics, virology, molecular biology, and other related disciplines. This thasis involves the research work of the preparation and cell imaging of two-photon nucleic acids fluorescent probes, including DNA and RNA probes. On the basis of the preliminary work, this chapter design synthesize and characterize the two-photon RNA fluorescence probes, explore the inherent rules that cationic organic molecules specially recognize RNA and the influence of molecular configuration on specific recoganize ability, master the regulation rules of RNA probes, and eventually reach the targets of producing high-performance nucleic acids probes. Several classes of molecular probes have been developed for RNA detection in living cells, including (a) ODN probes;(b) linear fluorescence resonance energy transfer (FRET) probes;(c) dual-labeled oligonucleotide hairpin probes (e.g., molecular beacons);(d) dual FRET molecular beacons;(e) autoligation probes; and (f) probes using fluorescent proteins as reporters. But, because many RNA fluorescent probes do not possess membrane-permeability, in order to obtaining fluorescent imaging on RNA in a living cell, they have to be injected into a living cell of target by microinjection. a technology that is destructive to intact cells and interferes with biological functions of cells. Therefore, it is very essential to develop a membrane-permeable, low-molecular-weight two-photon fluorescent probe for imaging RNA in living cells.In the third chapter, indole-based mono-cationic salts (INR1and INR2) have been synthesized and characterized successfully. According to their spectral response to RNA in vitro and fluorescent imaging in four different living cell lines (SiHa, HeLa, PC3and MDA-MB-231), we identify INR1and INR2as RNA-selective fluorescent turn-on probes with large two-photon excitation fluorescence action absorption cross-sections (Φxδ) when binding to RNA. And two dyes also have very good membrane permeability and can image RNA in nucleoli and cytoplasm in living cells by confocal and two-photon fluorescence microscopy (TPM). Furthermore, they possess good counterstain compatibility with Hoechst33342, a classic DNA-staining dye for live cells, which is very important to RNA-DNA colocalization.In the forth chapter, A new carbazole-derived dicationic compound, namely2,7-bis(1-hydroxyethyl-4-vinylpyridinium iodine)-N-ethylcarbazole (HVC-6) has been obtained. Moreover, it possesses the potential of imaging RNA in nucleoli and cytoplasm in two-photon fluorescence microscopy and exhibits good counterstain compatibility with the commercial fluorescent nucleic dye DAPI.In the fifth chapter, we have focused on small-molecule-based probes capable of imaging intracellular target. This chapter have designed a LMW mitochondrial probe,3,5-bis((E)-2-(pyridin-4-yl)vinyl)-1H-indole monoiodide (INSC12) based on the conjugation of the indole fluorophore with an anchor group. This anchor group is composed of long alkyl chains and one zwitterionic group, allowing strong interaction with the lipid membrane. Consequently, the probe INSC12exhibits a process for imaging cell membranes, rapidly entering the cell and lighting up the mitochondria; Moreover, the probe possesses some ability for the rapid detection and tracking of morphological change and for observing the dynamical behavior of mitochondria over a long period of time with appreciable tolerance of microenvironmental change.In summary, a series of highly selective fluorescent probes for cell membrane (HVC-12/HVC-18), RNA (INR1/INR2/HVC-6) and mitochondrial (INSC12) detection have been designed and synthesized successfully. In particular, this thesis first achieve live-cell membrane and deep-tissue imaging of enrich Lo phase based aggregation-induced emission (AIE) mechanism. In addition, INR1/INR2and HVC-6also achieve RNA detection used confocal fluorescence microscopy fluorescent microTPM; Meanwhile, the recognition mechanism between INR1/INR2and RNA and their two-photon property have been studied in detail. This thesis will lay the foundation to get the commercialization of RNA/cell membrane fluorescent probes. |
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