Yuming Sun

Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells.

The synthesis and characterization of a pair of novel pyridine cation derivatives possessing two-photon excitation fluorescence (TPEF) properties and selectively staining mitochondria in living SiHa cells within 30 min, CAI and CAEI, were reported. And the green-emitting CAI displayed a much larger two-photon excitation fluorescence action absorption cross-section (δ × Φ) of 328 g at 860 nm in comparison with commercial MitoTracker Green (MTG) with maximum δ × Φ value of 2.18 g at 850 nm. As is known to all, δ × Φ is a crucial parameter to obtain a high-quality microscopic photo in living cells in two-photon microscopy (TPM). Moreover, the fact that the co-localization coefficient between CAI and conventional MitoTracker Red (MTR) was 0.95 in SiHa cells demonstrated specific staining performance of CAI to mitochondria. As biosensors, both CAI and CAEI possessed a number of beneficial properties such as large δ × Φ and Stokes shifts, good membrane permeability, long retention time, high photostability and excellent counterstain compatibility with different biosensors for instance Hoechst 33342 and D 307, which ranked them as one of the best TPEF mitochondrial probes. Furthermore, related mechanism research suggested that their localization properties were dependent on the mitochondrial membrane potential in living cells. And their remarkable properties can extend the investigation on mitochondria in a biological context.

Low molecular weight fluorescent probes with good photostability for imaging RNA-rich nucleolus and RNA in cytoplasm in living cells.

We have synthesized two low molecular weight organic molecules, PY and IN successfully, which selectively stain nucleolus and cytoplasm of living cells in 30 min, with a much lower uptake in the nucleus. Nucleic acids electrophoresis and digest test of ribonuclease indicate their markedly higher affinity for RNA, especially PY. Moreover their RNA localization in cells is further supported by digest test of ribonuclease, namely, the nucleolar fluorescence signal is distinctly lost upon treatment with RNase. And, the fact that live cells stained by PY and IN still possess physiological function can be confirmed: 1) MTT assay demonstrates that the mitochondria of cells stained remains its electron mediating ability, 2) Double assay of PY/IN and propidium iodide as well as trypan blue testing show that the membrane of cells stained still is intact. Importantly, compared with the only commercial RNA probe, SYTO RNA-Select, PY and IN exhibit much better photostability when continuously illuminated with 488 nm laser and mercury lamp. These results prove that PY and IN are very attractive staining reagents for visualizing RNA in living cells.

Fluorescent imaging of acidic compartments in living cells with a high selective novel one-photon ratiometric and two-photon acidic pH probe.

Fluorescent imaging of acidic compartments in living cells was carried out successfully by a novel molecule (CAE) that contained a pyridine unit and a carbazole core. As the protonation of the pyridine N atom of CAE, pH-dependent absorption and fluorescence properties were shown with a pKa of 5.47 which matches the pH range of intracellular acidic compartments. Moreover, a large Stokes shift, a significant enhancement in ratios of I544 nm/I460 nm and a distinct two-photon turn-on character were exhibited in spectral analysis. Meanwhile, direct intracellular imaging and standard double-staining experiments of CAE and LTR (co-localization coefficient: 0.83) revealed that CAE is an effective one-photon ratiometric and two-photon acidic pH probe for imaging intracellular acidic compartments. The pH distribution pattern of intracellular acidic compartments can be obtained facilely by CAE. In especial, CAE possessed well membrane-permeability, brilliant selectivity among various bioanalyte and excellent counterstain compatibility with Hoechst 33342, MTR and LTR.

Red-Emitting Mitochondrial Probe with Ultrahigh Signal-to-Noise Ratio Enables High-Fidelity Fluorescent Images in Two-Photon Microscopy.

Herein, we reported a red-emitting probe (E)-4-(2-(8-hydroxy-julolidine-9-yl)vinyl)-1-methylpyridin-1-ium iodide (HJVPI) on a rotor mechanism with an ultrahigh signal-to-noise ratio. HJVPI could give high-fidelity fluorescent images of mitochondria in living immortalized and normal cells and be suitable for IR excitation source of two-photon microscopy and various excitation sources of confocal microscopy. As a rotor, its single/two-photon fluorescence intensities directly depended on environmental viscosity. And, as a mitochondrial probe, it displayed much larger two-photon absorption cross sections in comparison with commercial MitoTracker Green FM and MitoTracker Red FM. Moreover, the fact that living cells stained by HJVPI still possessed physiological function could also be confirmed: (1) MTT assay demonstrated that the mitochondria of cells stained retained their electron mediating ability and (2) double assay of HJVPI and SYTOX Blue nucleic acid stain (S-11348) showed that the plasma membrane of the cells stained was still intact. In addition, HJVPI possessed a number of beneficial properties in bioimaging such as good membrane permeability, high photostability, and excellent counterstain compatibility with Hoechst 33342. Related mechanism research suggested that its localization property was dependent on the mitochondrial membrane potential in living cells. All its remarkable properties can extend the investigation on mitochondria in a biological context.

Electrochemiluminescence resonance energy transfer between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor and its biological application

Novel electrochemiluminescence resonance energy transfer (ECRET) between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor is investigated. The ECRET technique can be used to study the interactions and conformational changes of proteins.

A membrane-permeable dye for living cells with large two-photon excited fluorescence action cross-sections for bioimaging

The development of two-photon fluorophores remains an important issue. Dyes that possess both large two-photon excited fluorescence action cross-sections and cell membrane permeability are especially in demand to maximize the underlying virtue of two-photon microscopy for bioimaging. Herein, a novel two-photon excited fluorescence dye has been synthesized. This V-shaped dye exhibited large two-photon excited fluorescence action cross-sections and high plasma membrane permeability. Cell imaging experiments demonstrated that the dye could stain living cells with bright two-photon excited fluorescence. All the results have indicated the potential of the dye as a basic platform for the development of two-photon excited fluorescence probes.

A two-photon ratiometric fluorescent probe enables spatial coordinates determination of intracellular pH

We reported a two-photon ratiometric fluorescent probe for detecting intracellular pH. When excited with 800 nm laser, an optimal output of laser as the routine equipment of two-photon fluorescence microscopy, the two-photon excited fluorescence of this probe showed distinct emission peak shift as large as 109 nm upon the change of pH values in vitro. Very importantly, the experiment results show that this probe has large two-photon absorption cross-section at pH 4.5 at 800 nm of 354 g, which ranks it as one of the best two-photon ratiometric fluorescent pH probes, and its working pH value is between 4.0 and 8.0 which could fit the intracellular pH range. Moreover, utilizing this probe, the two-photon ratiometric fluorescent images in living cells have been obtained, and the spatial coordinates of intracellular pH can be mapped. At the same time, the probe also exhibited selectivity, photostability and membrane permeability. And the photophysical properties of this probe in various solvents indicated that these photophysical properties variations are due to an intramolecular charge transfer process. At last, the imaging depth of the probe in liver biopsy slices was investigated. The experimental results demonstrated the maximum imaging depth can arrive 66 µm in living rat liver tissues.

Phospholipid-Biomimetic Fluorescent Mitochondrial Probe with Ultrahigh Selectivity Enables In Situ and High-Fidelity Tissue Imaging

In situ and directly imaging mitochondria in tissues instead of isolated cells can offer more native and accurate information. Particularly, in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy, it is a routine examination item to directly observe mitochondrial morphology and number in muscle tissues from patients. However, it is still a challenging task because the selectivity of available probes is inadequate for exclusively tissue imaging. Inspired by the chemical structure of amphiphilic phospholipids in mitochondrial inner membrane, we synthesized a phospholipid-biomimetic amphiphilic fluorescent probe (Mito-MOI) by modifying a C18-alkyl chain to the lipophilic side of carbazole-indolenine cation. Thus, the phospholipid-like Mito-MOI locates at mitochondrial inner membrane through electrostatic interaction between its cation and inner membrane negative charge. Simultaneously, the C18-alkyl chain, as the second targeting group, is deeply embedded into the hydrophobic region of inner membrane through hydrophobic interaction. Therefore, the dual targeting groups (cation and C18-alkyl chain) actually endow Mito-MOI with ultrahigh selectivity. As expected, high-resolution microscopic photos showed that Mito-MOI indeed stained mitochondrial inner membrane. Moreover, in situ and high-fidelity tissue imaging has been achieved, and particularly, four kinds of mitochondria and their crystal-like structure in muscle tissues were visualized clearly. Finally, the dynamic process of mitochondrial fission in living cells has been shown. The strategy employing dual targeting groups should have reference value for designing fluorescent probes with ultrahigh selectivity to various intracellular membranous components.

Butyrate promotes visceral hypersensitivity in an IBS‐like model via enteric glial cell‐derived nerve growth factor

Altered visceral sensation is common in irritable bowel syndrome (IBS) and nerve growth factor (NGF) participates in visceral pain development. Sodium butyrate (NaB) could induce colonic hypersensitivity via peripheral up‐regulation of NGF in animals. Enteric glial cells (EGCs) appear to be an important source of NGF. Whether butyrate could induce visceral hypersensitivity via increased EGC‐derived NGF is still unknown.

Prospective study evaluating the effect of mifepristone on E-cadherin expression in villi in early pregnancy

BACKGROUND E-cadherin plays an important regulatory role in implantation, embryo development and placentation. This study aimed to determine the effect of mifepristone on E-cadherin expression in human villi in early pregnancy. STUDY DESIGN Forty healthy women seeking elective pregnancy termination at 5-7 weeks of gestation were recruited. Of these, 22 women chose medical termination (mifepristone-treated group) and took 25mg mifepristone every 12h for 3 days and 600μg buccal misoprostol on the morning of the fourth day. The other 18 women underwent vacuum aspiration (control group). Following collection of villi, E-cadherin protein expression was assessed by immunohistochemical analysis, and E-cadherin mRNA expression was assessed by reverse transcription-polymerase chain reaction. RESULTS E-cadherin protein expression was significantly higher (p<0.05) in villous cytotrophoblast cells in the mifepristone-treated group compared with the control group. E-cadherin mRNA expression was also significantly higher (p<0.01) in the mifepristone-treated group compared with the control group. CONCLUSION E-cadherin expression in villi may be involved in mifepristone-induced pregnancy termination.