Zhen-Li Huang

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein-protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 degrees C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 degrees C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein-protein interactions at 37 degrees C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein-protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein-protein interactions in living cells and offer the potential to visualize protein-protein interactions in living animals.

Novel heterocycle-based organic molecules with two-photon induced blue fluorescent emission

Two-photon absorption and two-photon induced blue emission characteristics of a series of heterocycle-based organic molecules are investigated experimentally and by quantum-chemical computations. The molecules consist of a typical A–π–A′ structure, where heterocycle, styryl and formyl groups are employed as A, π-conjugated and A′ moieties, respectively. Experimental results indicate that significant enhancements in the blue emission efficiency and two-photon absorption cross-sections can be achieved by replacing S and O atoms with an N atom in the heterocycle acceptor moiety, which is also supported by the quantum-chemical computations. Additionally, larger two-photon absorption cross-sections can be obtained by choosing appropriate solvents, as indicated by the computations.

Two-photon absorption of a series of V-shape molecules: the influence of acceptor's strength on two-photon absorption in a noncentrosymmetric D–π–A–π–D system

A series of V-shape D–π–A–π–D molecules were synthesized based on pyrimidine as the acceptor. The strength of the acceptor (A) in these molecules was systematically adjusted through changing the substituent group in pyrimidine. Their two-photon absorption properties were measured by two-photon induced fluorescence techniques with a mode-locked Ti:sapphire laser (100 fs, 80 MHz) in the range of 720 to 880 nm. The results indicate that the strength of the acceptor has an important influence on the two-photon absorption properties of this noncentrosymmetric D–π–A–π–D system, and a molecule with a strong acceptor is liable to display a large two-photon absorption cross-section.

Modification of CdTe quantum dots as temperature-insensitive bioprobes

CdTe quantum dots (QDs) were synthesized in aqueous solution with 3-mercaptopropionic acid (MPA) as the stabilizer. The photoluminescence (PL) of CdTe QDs (3.5nm) is found to be temperature-dependent: as the temperature arising from 278K to 323K, the PL intensity declines to 50.2% of its original and PL emission peak shows obvious red-shift ( approximately 7nm). After modification of the QDs surface with denatured ovalbumin, the PL is more temperature-insensitive than before. The PL intensity retains more than 70% of its original and the emission peak shows less red-shift ( approximately 2nm). Moreover, it is found that the PL intensity and wavelength of denatured ovalbumin coated CdTe QDs are reversible during heating (323K)-cooling (278K) cycles. All the studies provide an important theoretical basis for searching temperature-insensitive bioprobes.

Frequency up-conversion of s-triazine derivatives via two photon absorption and second-harmonic generation

A three-branched octupolar compound, 2,4,6-tris[2-(4-N,N-diethylamino)phenylethenyl]-1,3,5-s-triazine (compound III), as well as its one- and two-branched analogues (compounds I and II respectively) have been synthesized. The structures of I and II have been determined by X-ray diffraction. The linear and nonlinear spectra of these compounds regularly vary: (1) the spectral positions (λmax values) of the linear absorption, the single-photon excited fluorescence (SPEF) and the two-photon excited fluorescence (TPEF) show regular red-shifts when the branch number increases from 1 to 3; (2) the spectral intensities, including the linear absorbance emax and the two-photon absorption cross-section σ, the SPEF and the TPEF intensities are all increased significantly when going from I to III. The values of emax/MW and σ/MW (of compounds I, II and III) vary with the ratios of 1.0 : 1.3 : 1.7 and 1.0 : 1.4 : 1.3, respectively. All the photophysical data indicate that by incorporating more branches into the triazine ring, the linear and nonlinear optical responses have been significantly enhanced, and that the spectral behaviour of II is closer to that of III, while more different from that of I. The single-branched compound I crystallizes in the noncentrosymmetric polar space group Cc. Its crystalline powder sample shows remarkable macro second-order nonlinear response with a SHG intensity of 46.8 times that of urea under a fundamental picosecond Nd : YAG laser beam (1064 nm). An important factor of such a large SHG intensity is that all the molecular dipoles take the same-oriented packing style that makes the crystal maximally polarized, fulfilling the optimization in the sense of crystal engineering.

Synthesis and structure–photophysical property relationships for two coumarinyl-based two-photon induced fluorescent molecules

Abstract The synthesis and the structure–photophysical property relationships for two new coumarinyl-based organic heterocyclic molecules are presented. These molecules consist of a typical A–π–D or A–π–A′ structure, where 3-carbonylcoumarinyl, vinyl and anthryl (or indolyl) groups are employed as acceptor (A), π-conjugated center (π) and donor (D) (or the other acceptor, A′) moieties, respectively. Based on steady-state as well as time-resolved techniques, it is found that this new kind of π-conjugated framework consisting of a 3-carbonylcoumarinyl acceptor and a vinyl π-conjugated center is a useful conjugated π-electron building block with potential applications in forming new kind of effective organic two-photon induced fluorescence (TPIF) molecules. Additionally, experimental results confirm that remarkable enhancements in the fluorescence quantum yield and TPIF activity can be obtained by replacing anthryl group with an indolyl group in the terminal moiety of the coumarinyl-based π-conjugated framework.

Quantum dot optical encoded polystyrene beads for DNA detection

A novel multiplex analysis technology based on quantum dot (QD) optical encoded beads was studied. Carboxyl functionalized polystyrene beads, about 100 microm in size, were precisely encoded by the various ratios of two types of QDs whose emission wavelengths are 576 and 628 nm, respectively. Then the different encoded beads were covalently immobilized with different probes in the existing of sulfo-NHS and 1-[3-(Dimethylamino) propyl]-3-ethylcarbodiimide methiodide, and the probe density could reach to 3.1 mmol/g. These probe-linked encoded beads were used to detect the target DNA sequences in complex DNA solution by hybridization. Hybridization was visualized using fluorescein isothiocynate-labeled DNA sequences. The results show that the QDs and target signals can be obviously identified from a single-bead-level spectrum. This technology can detect DNA targets effectively with a detection limit of 0.2 microg/mL in complex solution.

A feasible and quantitative encoding method for microbeads with multicolor quantum dots.

Multicolor encoded beads were achieved by incorporating two color core-shell quantum dots (QDs) (CdSe/ZnS) to commercial polystyrene (PS) beads. By controlling the concentration ratios of the two quantum dots (QDs) in doping solutions, a series of codes with different intensity ratios were obtained. Based on the multiple encoded carboxylic modified polystyrene beads, fluorescent dyes labeled antibodies were distinguished successfully on the beads’ surface. It suggests that the encoded beads from this method have the practicability in biological applications and chemical analysis.

A Feasible Add-On Upgrade on a Commercial Two-Photon FLIM Microscope for Optimal FLIM-FRET Imaging of CFP-YFP Pairs

Fluorescence lifetime imaging microscopy (FLIM) based on time-correlated single photon counting (TCSPC) is a widely used method for fluorescence resonance energy transfer (FRET). Here we report a feasible add-on approach to upgrade a commercial two-photon FLIM microscope into a single-photon FLIM microscope which provides optimal FLIM-FRET imaging of FRET pairs consisting of cyan fluorescent proteins (CFPs) as the donor and yellow fluorescent proteins (YFPs) as the acceptor. The capability of the upgraded system is evaluated and discussed, and the imaging performance of the system is demonstrated using FLIM-FRET experiments with a representative CFP-YFP FRET pair (mCerulean-mCitrine).

A feasible method for comparing the power dependent photostability of fluorescent proteins

A feasible method of combining the concept of fluorescence half-life and the power dependent photobleaching rate for characterizing the practical photostability of fluorescent proteins (FPs) was introduced. Furthermore, by using a fluorescent photostability standard, a relative comparison of the photostabilty of FPs from different research groups was proposed, which would be of great benefit for developing novel FPs with optimized emission wavelength, better brightness, and improved photostability. We used rhodamine B as an example to verify this method and evaluate the practical photostability of a far-red FP, mKate-S158C. Experimental results indicated good potential of this method for further study.