Minggang Tian

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.

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.

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.

Lighting up cysteine and homocysteine in sequence based on the kinetic difference of the cyclization/addition reaction.

A novel one- and two-photon fluorescent probe CB1 has been developed for discriminating Cys and Hcy in a successive manner with high selectivity. The discrete time-dependent fluorescent responses enable us to sequentially detect Cys and Hcy in different time windows. Two-step reaction and kinetic modes were used to explain the sensing mechanism. As a promising biosensor for cell imaging, CB1 has been confirmed to exhibit membrane permeability to intact cells, low cytotoxicity to viable cells and photostability to ultraviolet light excitation. Furthermore, the results from the control assay have shown that the one- and two-photon fluorescence of CB1 within cells is associated with intracellular mercapto biomolecules but yet there is little interference with physiological pH value, viscosity and common bioanalytes. Finally one- and two-photon fluorescent images of CB1 within living SiHa cells have been presented.

Spatially Dependent Fluorescent Probe for Detecting Different Situations of Mitochondrial Membrane Potential Conveniently and Efficiently

The feedback from mitochondrial membrane potential (MMP) in different situations (normal, decreasing, and vanishing) can reflect different cellular status, which can be applied in biomedical research and diagnosis of the related diseases. Thus, the efficient and convenient detection for MMP in different situations is particularly important, yet the operations of current fluorescent probes are complex. In order to address this concern, we presented herein a spatially dependent fluorescent probe composed of organic cationic salt. The experimental results from normal and immortalized cells showed that it 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. According to the cell morphology, there is a straightforward spatial boundary between the nucleus and cytoplasm where mitochondria locate; thus, the three situations of MMP can be point-to-point indicated just by fluorescence images of the probe: that all probes accumulate in mitochondria corresponds to normal MMP; that probes locate both in the mitochondria and nucleus corresponds to decreasing MMP; that probes only target the nucleus corresponds to vanishing MMP. It is worth noting that counterstaining results with S-11348 indicated that the spatially dependent probe could be applied to distinguishing dead from viable cells in the same cell population. Compared with the commercial Cellstain-Double staining kit containing calcein-AM and propidium iodide (PI), this probe can address this concern by itself and shorten the testing time, which brings enormous convenience for relevant researches.

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.

4-tert-butylphenoxy substituted phthalocyanine with RGD motif as highly selective one-photon and two-photon imaging probe for mitochondria and cancer cell

An unsymmetrical phthalocyanine based one- and two-photon fluorescence imaging probe that substituted with 4-tert-butylphenoxy and RDGyK moieties was developed and characterized by UV-vis and high-resolution MALDI-TOF/MS. The conjugate is non-aggregated in N,N-dimethylformamide, with relatively weak fluorescence emission (ΦF = 0.023) and high singlet oxygen quantum yield (ΦΔ = 0.55). Conjugation of the cyclic peptide sequence c(RGDyK) can enhance the cellular uptake towards the DU145 and PC3 cells. While the fluorescence is greatly enhanced in mitochondria, the conjugate is non-cytotoxicity either in dark or upon exposure to red-light with dose up to 12 J.cm−2. The results suggest that this conjugate is a promising multifunctional imaging probe for mitochondria and cancer.