| Chapter 1:This chapter explained the advantages of fluorometry with the combination of confocal laser scanning microscopy and two-photon imaging technology, the basic structure and performance of cyanine dyes; highlights the research progress of cyanine dyes on intracellular pH and biothiols in the recent years. Based on the review of previous work, we presented the design idea and the research content of this paper.Chapter 2:A novel ratiometric emission fluorescent probe, 1,1-dimethyl-2-[2-(quinolin-4-yl)vinyl]-1H-benzo[e]indole (QVBI), is facilely synthesized via ethylene bridging of benzoindole and quinoline. The probe exhibits ratiometric fluorescence emission (F522nm/^630nm) characteristics with pKa 3.27 and linear response to extreme-acidity range of 3.8-2.0. Also, its high fluorescence quantum yield (0=0.89) and large Stokes shift (110 nm) are favorable. Moreover, QVBI possesses highly selective response to H* over metal ions and some bioactive molecules, good photostability, and excellent reversibility. The probe has excellent cell membrane permeability and is further applied successfully to monitor pH fluctuations in live cells and imaging extreme acidity in Escherichia coli cells without influence of autofluorescence and native cellular species in biological systems.Chapter 3:We synthesize a novel pH fluorescent probe, 1,1-dimethyl-2-((E)-2-(pyridin-4-yl)vinyl)-1Hbenzo[e]indole (PVBI), for imaging in living cells via ethylene bridging of indole derivatives and pyridine. The pH titration indicated that the probe exhibits sensitive pH-dependent behavior with pKa 3.87 and responded linearly and rapidly to minor pH fluctuations within the range of 3.0-4.6 (linear coefficient of 0.9968). Thus PVBI could be potentially useful for quantitative determination of pH within the acidic window. Also, PVBI displays a notably large Stokes shift of 93 nm which could effectively reduce the excitation interference. Moreover, PVBI possesses highly selective response to H+over various metal ions, good photostability and excellent reversibility. The confocal laser scanning microscope images for PVBI diffusing into cells in real-time and detection of pH in living cells were achieved successfully, suggesting the probe has excellent cell membrane permeability and could be applied to monitor pH fluctuations in living cells.Chapter 4:A novel fluorescent probe for extreme acidity is developed using biocompatible indole derivatives as the fluorophore. The probe is linearly proportional to extreme acidity range of 2.3-3.6 with pK & 2.89. Moreover, it displays promising spectroscopic features such as large Stokes shift (145 nm), highly selective response to H+, good photostability and excellent reversibility. The notable color change from yellow to pink with decreasing pH make it can also serve as a "naked-eye" fluorescence indicator for acidic pH. The confocal laser scanning microscope images display the probe possesses excellent cell membrane permeability, and is further applied successfully to imaging extreme acidity in E. coli cells.Chapter 5:A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed on the basis of a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylate-functionalized merocyanine probe shows not only a mitochondria-targeting property but also selectively marked blue-to-green emission change upon detecting/reacting with Cys over other biothiols such as homocysteine (Hey) and glutathione (GSH) and hydrogen sulfide (H2S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F518/F452), which are linearly proportional to Cys concentrations in the range of 0.5-40 ?M. More importantly, the-probe and its released fluorophore, merocyanine, exhibit strong two-photon excited fluorescence (TPEF) with two-photon action cross-section (??max) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively, which is highly desirable for high contrast and brightness ratiometric two-photon fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150 ?m by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.Chapter 6:We synthesize a Turn-on fluorescent probe (MCY-NBD) for sensing biothiol. MCY-NBD could high sensitively and rapid response (2-min) to biothiol near physiological pH range (6.0-6.0), and the fluorescence intensity increase about eight to nine times. Moreover, it displays good photostability and highly selective response to biothiol. The excellent optical performance is expected to be applied to selectively detect biothiol in living biological systems. |
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