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The effects of amine donors (a:NH 2 , b:NMe 2 , c:NMePh, d:NPh 2 ) and conjugation length on the molecular hyperpolarizabilities of a series of dipolar molecules have been theoretically investigated by using CPHF/6-31G method. The first hyperpolarizabilities (β) of p-nitrobenzene derivatives increase with the donor in the order, NH 2 < NMe 2 < NMePh < NPh 2 , whereas slightly different order is observed in more conjugated derivatives, i.e., NH 2 < NPh 2 < NΜe 2 < NMePh. The result has been attributed to the extent of charge transfer and torsion angle. Moreover, the results show that "non-traditional" π-conjugation effect exists in small compounds and decreases as the conjugation length between donor and acceptor increases.

The first synthesis of phenylacetylene macrocycle with large two-photon absorption cross section is reported.

Novel two-dimensional octupoles containing azo groups and their oligomers have been synthesized and their first hyperpolarizabilities were determined. The λmax and β(0) values of azo octupoles are nearly the same regardless of the nature of the bridge or the conjugation length. On the other hand, 1,3,5-tricyano-2,4,6-tris[4-(p-diethylaminostyryl)styryl]benzene has a larger β(0) value than the corresponding azo analogue probably due to the more efficient conjugation ability of CC compared with the NN bond. The β(0)/MW of the octupoles are larger than the corresponding dipoles, whereas those of the azo octupoles are comparable to the latter. Also, retention of monomeric β(0) is observed in the octupolar oligomers.

The G-quadruplexes play a biological role in DNA telomere ends,the purine-rich DNA strands of the oncogenic promoterelements such as c-myc and c-kit, and RNA 5'-untranslatedregion (UTR) in close to translational start sites. Telomereslocated at the ends of eukaryotic chromosomes are com-posed of the tandem DNA repeats of guanine-rich sequencesand essential for chromosome stability. They appear to playa critical role in cellular aging and cancer.

1,3,5-Tricyano-2,4,6-tris[2-(thiophen-2-yl)vinyl]benzene derivatives have been synthesized as a new type of octupolar nonlinear optical molecules and their linear and nonlinear optical properties were determined. The λ m a x values increased with the conjugation length and as the conjugation bridge was changed from phenyl to thienyl group. For phenyl and thiophene derivatives, the β value increased with conjugation length. In contrast, the b values of the thiophene derivatives were much smaller than the corresponding phenyl derivatives.

E-mail: brcho@cju.ac.krReceived June 27, 2011, Accepted August 9, 2011Key Words : G-rich RNA, RNA aptamer, RNase T1, Nuclease S1, RNase V1 RNA structure has sometimes been analyzed by thebiophysical method of NMR (nuclear magnetic resonance),which has not been popular because the RNA with smallsize only is available and a large amount of RNA is requiredfor the structural analysis with NMR.

Copper ions play a crucial role in living systems as cofactors of numerous metalloenzymes. To quantitatively estimate the Cu(2+) concentration in human tissue, we have developed a two-photon (TP) probe with an internal reference (ACCu2) that shows significant TP action cross-section and high selectivity for Cu(2+) and can quantitatively estimate the Cu(2+) concentration in human colon tissues by dual-color two-photon microscopy (TPM) imaging with minimum interference from other competing metal ions or pH and minimum cytotoxicity and photostability problems. The Cu(2+) concentrations in human normal colon, polyp, and colon cancer tissues were found to be 8.3 ± 0.3, 13 ± 2, and 22 ± 3 μM, respectively. This result suggests that ACCu2 may be useful for the diagnosis of human colon cancer.

We have developed TP probes for [Zn2+]cyto and [Zn2+]mito, which emit TPEF at widely-separated wavelength regions. The new probes can simultaneously detect [Zn2+]cyto and [Zn2+]mito in live cells, as well as in living tissues by dual-color TPM imaging.

We summarize the nonlinear optical (NLO) properties of octupolar molecules, crystals, and films developed in our laboratory. We present the design strategy, structure-property relationship, and second-order NLO properties of 1,3,5-trinitro- and 1,3,5-tricyano-2,4,6-tris(p-diethylaminostyryl)benzene (TTB) derivatives, TTB crystals, and films prepared by free-casting TTB in poly(methyl methacrylate) (PMMA). The first hyperpolarizability of TTB was fivefold larger than that of the dipolar analogue. Moreover, the TTB crystal showed unprecedentedly large second-harmonic generation (SHG). While TTB crystal films (20 wt% TTB/PMMA) on various substrates showed appreciable SHG values, the cylinder film exhibited much larger SHG values and large electro-optic (EO) coefficients. The large SHG values and EO coefficients, as well as the high thermal stability of the cylinder film, will make it a potential candidate for NLO device applications.

Stilbene and heteroaromatic stilbene derivatives have been synthesized and their β values were determined. The result of HRS measurements indicates that the β(O) values are larger for the heteroaromatic stilbenes than the stilbenes. For stilbene derivatives, the β(O) increased as the acceptor strength was increased.

Secondary structure models of Kin.46 ribozyme and the tethered ribozyme derived from Kin.46 (15-nt tether shown). Paired elements P1 through P4, connecting loop L3, and joining elements J1/2, J2/3, J3/2, J2/1 and J1/4 are indicated. The tethered ribozyme is built from the two strands, outer strand and inner strand. Thick line in the tethered ribozyme indicates 13-nt DNA oligonucleotide com-plementary to the 15-nt tether.

Secondary structures of two ribozyme versions derived from the Kin.46 self-thiophosphorylating ribozyme by the internal truncations. (A) A ribozyme119 derivative has 4 strands; a 7-mer, a upper (up) strand, a lower (lw) strand and an activating oligomer (AO)with 18 nucleotide. (B) A ribozyme103 derivative also has four diffe-rent strands but the length of a up strand and an lw strand is short. Only RNA portions at the RNA-RNA double strand region in a up strand were substituted for DNA portions.

A new level shifter using low‐temperature polycrystalline silicon (poly‐Si) thin‐film transistors (TFTs) for low‐power applications is proposed. The proposed level shifter uses a capacitive‐coupling effect and can reduce the power consumption owing to its no‐short‐circuit current. Its power saving over the conventional level shifter is 72% for a 3.3 V input and a 10 V output.

The two-photon absorption (TPA) cross section and the second hyperpolarizability (γ) of the linear quadrupolar molecule are theoretically investigated by using the ab initio calculation methods for a few series of quadrupolar molecules. The relationships between the molecular structure and the TPA as well as the NLO property are established. It is found that, as the strengths of donors and acceptors increase, (i) both the energy gap between the first excited electronic state, |e>, and the ground electronic state, |g>, and that between the TPA-allowed excited electronic state |f> and |g> decrease, (ii) the energy gap difference, |Efe − Eeg|, which is the detuning factor affecting the TPA matrix element, decreases, and (iii) the product of the transition dipole matrix elements, | |, associated with the TPA increases. These effects are combined to make the TPA cross section monotonically increase as the donor and acceptor strengths increase. The second hyperpolarizabilities of the same series of quadrupolar ...

The mechanism of polymerization reaction of 2,5-bis(tetrahydrothiopheniomethyl)furan dichloride was studied kinetically. The reaction proceeds by the (E1cb)irr mechanism to afford a 2,5-dihydrofuran intermediate M, which undergoes a free radical polymerization reaction. Comparison of the kinetic results reveals that the rate of elimination reaction increases, whereas that for the subsequent polymerization reaction decreases as the aromatic moiety of the bis(sulfonium) salts is changed from phenyl to thiophene to furan. The results have been attributed to the decreased resonance energy of the aromatic rings.

Second-order optical nonlinearities of a series of dipolar photoconductive chromophores have been investigated by semiempirical quantum chemical calculations using time-dependent Hartree–Fock formalism in conjunction with PM3 parameter and experimental evaluation of the first molecular hyperpolarizability ( β ). A linear correlation between the measured and calculated β values was observed, and the structure–property relationship discussed in terms of the structural variation. It was noted that the optical nonlinearities of the dipolar photoconductive molecules with carbazole, indole, or indoline as donor units are large enough for the electrooptic and photorefractive applications.

A-Si layer was crystallized at a temperature of 400 °C in the presence of a modest electric field of 100 V/cm. An accumulation of needlelike crystallites in the crystallized poly-Si has been found after crystallization. This results from the migration of NiSi2 precipitates through a-Si. The field effect mobility and threshold voltage for a SMC poly-Si TFT using a maximum process temperature of 400 °C were 76 cm2/Vs and 0.8 V, respectively. The performance of a SMC poly-Si TFT is comparable to that of an ELA poly-Si TFT.

Elimination reactions of YC6H4CH2C(O)OC6H3-2-X-4-NO2 promoted by R2NH in MeCN have been studied kinetically. The reactions are second-order and exhibit Bronsted β = 0.52–0.87, |βlg| = 0.37–0.65, and ρH = 1.9–4.1. The β and ρH values decrease as the leaving group becomes less basic and the |βlg| values decrease with a stronger base. The results are interpreted in terms of an E1cb-like transition state with extensive Cβ–H bond cleavage, significant negative charge development at the β-carbon, and limited Cα–OAr bond cleavage. For all reactions, the Hammett plots are curvilinear and show downward curvature. The curvature is more pronounced as the leaving group becomes poorer. The results have been attributed to the highly carbanionic transition state, which is moved towards the reactant on the horizontal reaction coordinate by a stronger electron-withdrawing substituent.

The Kin.46 kinase ribozyme was selected for the transfer of the thiophosphate from ATP- γ-S to its own 5’ hydroxyl end in the presence of oligo-nucleotide effector which is complementary to its 3’ primer binding sequence (PBS) used in the ampli-fication steps during the original selection for acti-vity.

Ribozyme119 derivatives from the Kin.46 self-thiopho-sphorylating ribozyme by internal deletions has 3 strands; a left high (LH) strand, a lower (lw) strand and an activating oligomer (AO) with 18 nucleotide (A), and has 4 strands; a 7-mer, an upper (up) strand, a lower (lw) strand and an activating oligomer (AO) with 18 nucleotide (B). For a lw strand, dyes (Cy3 and Cy5) were incorporatedto the 5’-end during transcription with the class II promoter using AMP-dye-AMP as a primer. Dye-labeled AO was purchased.

The polymerization kinetics of 2,5-bis(tetrahydrothiopheniomethyl)thiophene dichloride were studied. The reaction proceeds via a 2,5-dihydrothiophene intermediate M. Results of kinetic and product studies revealed that the 1,6-elimination proceeded by an (E1cb)irr mechanism. The rate of the disappearance of M was not influenced by the addition of OH-, carbanions, and tetrahydrothiophene but was accelerated by S2O82- and inhibited by TEMPO. In addition, the reaction mixture exhibited an electron spin resonance signal with a g value of 2.003 G and induced the polymerization of acrylamide. These results indicated a free radical polymerization mechanism.

Enzymes and chemical which have mainlybeen used for probing RNA structure in solution, are double-strand-specific RNase V1, single-strand-specific nucleaseS1, RNase T1 which has a specificity for a guanine in singlestrand region, and kethoxal (3-ethoxy-1,1-dihydroxy-2-butanone), which modify the N1 and N2 of guanine in thesingle strand. Hydroxyl radical (·OH) has also been used forthe structural analysis of RNA. Exposed nucleotides aredamaged by hydroxyl radical while nucleotides involved intertiary contacts are protected from damage, making it afavorable approach for establishing exterior/interior relationsfor RNA.

The first evaluation of the β values and thermal stability for octupolar molecules derived from hexaazatriphenylene derivatives is reported.

Elimination reactions of (Z)-thiophene- and (Z)-furan-2-carbaldehyde O-benzoyloximes 1 and 2 with DBU in MeCN have been investigated kinetically. The reactions are second order and exhibit substantial values of Hammett ρ and kH/kD values, and an E2 mechanism is evident. The relative rates of elimination from (Z)-ArCHNOC(O)C6H4Y are 1/1.1/0.6 for Ar = phenyl/thienyl/furyl, respectively. For reactions of 1 with DBU in MeCN, kH/kD = 8.2 ± 0.1, Hammett ρ = 1.22 ± 0.19, β1g = −0.43 ± 0.01, ΔH⧧ = 5.9 ± 0.1 kcal/mol, and ΔS⧧ = −28.5 ± 0.3 eu have been determined. The corresponding values for 2 are kH/kD = 8.8 ± 0.2, ρ = 1.87 ± 0.05, β1g = −0.55 ± 0.10, ΔH⧧ = 6.5 ± 0.1 kcal/mol, and ΔS⧧ = −29.0 ± 1.5 eu. The kH/kD, Hammett ρ, and |β1g| values increase as the β-aryl group is changed in the order phenyl < thienyl < furyl. The results indicate that the transition state structure for the nitrile-forming elimination changes slightly toward product-like with the change of the β-aryl group.

The first evaluation of the β for 2-[2-(p-diethylaminophenyl)vinyl]furan derivatives is reported in comparison with the corresponding thiophene derivative.

Elimination reactions of (E)-2,4-dinitrobenzaldehyde O-aryloximes 1−3 promoted by EtO-, PhC(CH3)NO-/PhC(CH3)NOH, and CF3CH2O-/CF3CH2OH buffers in ethanol have been studied kinetically. The reactions produced 2,4-dinitrobenzonitrile and aryloxides as the only products. The observed second-order kinetics, Bronsted β = 0.55−0.75, |βlg| = 0.39−0.48 are consistent with the E2 mechanism. The Bronsted β decreased as the leaving group was made more nucleofugic and the |βlg| increased with a weaker base. The result can be described by a positive interaction coefficient, pxy = ∂β/∂pK1g = ∂β1g/∂pKBH > 0, which provides additional support for the E2 mechanism.

Polymerization reactions of α,α‘-bis(tetrahydrothiophenio)-p-xylene dichlorides with OH- in H2O have been studied kinetically. The reactions proceeded via the α-tetrahydrothiophenio-p-xylylene intermediates. Results of the H−D exchange experiments and kinetic studies reveal that the 1,6-elimination forming the intermediates proceeds reversibly via the ylide intermediate. The observed rate of disappearance of the intermediate was increased by the addition of OH-, tetrahydrothiophene, and S2O82- as well as by the photoirradiation with a tungsten lamp in the presence of Rose Bengal and was inhibited by TEMPO. However, the rate was found to be independent of ylide concentration. From these results, an elimination−free radical polymerization mechanism is proposed. The microscopic rate constants for each step of this mechanism were calculated from the change in the intermediate concentration with time. All of the rate data showed excellent correlations with the substituent constants in the Hammett plot.

ion from the ribose 4' carbon leads to strand scission. The cleavage reaction of the kinase ribozyme with hydroxyl radical was quenched with thiourea and the products were analyzed by PAGE. Hydroxyl radical cleavage reaction was performed both in the absence and the presence of ATP (Fig. 3). Sequence C88GGGG92 was cleaved more strongly in the presence of ATP than at the absence of ATP, suggesting that this sequence which is conserved in ATP aptamers and close to catalytic core, is exposed by ATP in the secondary structure model of kinase ribozyme molecule. In conclusion, the structure of kinase ribozyme was analyzed in solution with probes such as RNase T1, RNase V1, kethoxal and hydroxyl radical generated from Fe/ EDTA/H2O2. The result suggested that 1) helices P2 and P3 are stabilized in the presence of ATP, 2) nucleotides in loop J3/2 are exposed in the presence of ATP, and 3) single strand region L3, which does not play an important role in the activity of this kinase ribozyme, is also exposed in the secondary structure model. Experimental Section Preparation of RNA. The kinase ribozyme strand was synthesized by run-off in vitro transcription with T7 RNA polymerase from the DNA template to which the T7 promoter was annealed. The resulting RNA was treated with CIP (calf intestinal alkaline phosphatase) to remove 5' end Figure 2. RNase T1 cleavage sites of ribozyme labeled at the 5'end, following kethoxal modification. Kethoxal modification reaction was performed at the 50 mM Mg concentration in the absence and presence of 10 mM ATP. The cleaved guanines which are in double strand, are indicated by arrows. A and P indicate the absence and the presence of ATP, respectively. Notations are as described in the legend to Figure 1. Figure 3. Hydroxyl radical cleavage sites of ribozyme labeled at the 5'-end. The cleavage reaction was performed at the 50 mM Mg concentration in the absence and the presence of 10 mM ATP. The strong cleavages were occurred in the presence of 10 mM ATP, compare with the absence of ATP and their cleavage sites were denoted by bar. A and P indicate absence and presence of ATP, respectively. Notations are as described in the legend to Figure 1. Figure 4. Schematic representation of possible secondary structure of kinase ribozyme. Open arrows indicate the sites cleaved by RNase T1 at the absence of ATP, and stars and filled arrows indicate guanines (in the double) not modified with kethoxal and the sites cleaved by hydroxyl radical in the presence of ATP, respectively. 788 Bull. Korean Chem. Soc. 2006, Vol. 27, No. 5 Notes phosphate and then labeled at the 5' end using [γ-P]ATP and T4 polynucleotide kinase. Enzymatic cleavage reaction. 5'-terminal radiolabeled kinase ribozyme strand and activating DNA oligomer were heated in KCl/Pipes buffer (200 mM KCl in 150 mM PipesKOH, pH 7.0) at 90 C for 2 min and allowed to cool to RT (~21 C). These were adjusted to a final concentration of 50 mM MgCl2. Then 0.1-1 unit of nuclease S1 or 0.001-0.01 unit of RNase V1 or 0.1-1 unit of RNase T1 was added to the above mixture and then the reaction mixture was incubated for 20 min at RT. The cleavage products were recovered by ethanol precipitation and separated on a 12% polyacylamide gel in 90 mM Tris-borate (pH 8.3) and 2.5 mM EDTA containing 7 M urea. Kethoxal modification and RNase T1 cleavage reaction. 5'-end labeled RNA and activating DNA oligomer were renatured as above. The renatured mixture was adjusted to a final concentration of 10 mM (or 0 mM) ATP in the presence of 50 mM MgCl2. 1 uL of kethoxal was dissolve in 10 uL of 100% ethanol and 10 uL of 70% ethanol and 1-2 uL of dissolved kethoxal solution was added to the renatured mixture, which was incubated for 20-25 min at RT. The modified products were recovered by ethanol precipitation and then digested with RNase T1 for 15 min in the presence of 7 M urea. The cleavage mixtures were subject to a denaturing 12% polyacylamide gel electrophoresis. Fe2+/EDTA/H2O2 cleavage reaction. 5'-end labeled RNA and activating DNA oligomer were renatured as above. The renatured mixture was adjusted to a final concentration of 10 mM (or 0 mM) ATP in the presence of 50 mM MgCl2. 1 uL of 0.4 mM Fe(NH4)2(SO4)2·6H2O (freshly prepared), 1 uL of 0.8 mM EDTA, pH 8.0, 1 uL of 0.3% H2O2 and 1 uL of 1 mM DTT were added to the mixture and the reaction mixture was incubated for 5-10 min at RT. The reaction was quenched by adding thiourea to 1.3 mM and the cleavage products were recovered by ethanol precipitation and separated on a 12% polyacylamide gel containing 7 M urea. Acknowledgement. I appreciate Dr. Donald H. Burke (Department of Molecular Microbiology & Immunology and Biochemistry, University of Missouri) for his helpful discussion.

Elimination reactions of aryl esters of arylacetic acids 1 and 2 promoted by R2NH in MeCN have been investigated kinetically. The reactions are second-order and exhibit β = 0.44−0.84, |βlg| = 0.41−0.50, and ρH = 2.0−3.6. Bronsted β and |βlg| decrease with the electron-withdrawing ability of the β-aryl substituent. Hammett ρH values remain nearly the same, but the |βlg| value increases as the base strength becomes weaker. Both ρH and β decrease with the change of the leaving group from 4-nitrophenoxide to 2,4-dinitrophenoxide. The results are consistent with an E2 mechanism and a reaction coordinate with a large horizontal component corresponding to proton transfer. When the base−solvent system is changed from R2NH−MeCN to R2NH/R2NH2+−70 mol % MeCN(aq), the Bronsted β, ρH, and |βlg| decrease. Finally, the ketene-forming elimination reactions from p-nitrophenyl p-nitrophenylacetate promoted by R2NH/R2NH2+ buffers in 70 mol % MeCN(aq) have been shown to proceed by concurrent E2 and E1cb mechanisms.

Mitochondria provide the energy of the cells and are the primary site of oxygen consumption and the major source of reactive oxygen species. In mitochondria, metal ions and glutathione play vital roles in maintaining their structure and the redox environment. To understand their roles in mitochondria, it is crucial to monitor each of these chemical species in the mitochondria at the cell, tissue, and organism levels. An ideal tool for such purpose is the use of two-photon microscopy (TPM). Until recently, however, there has been no report on the two-photon (TP) probes suitable for such applications. In this paper, we summarize the mitochondria-targeted TP probes for Zn(2+), H(2)O(2), and thiols, as well as their bioimaging applications.

We provide an improved method for measuring the first order hyperpolarizability of organic nonlinear optical materials in solution by the hyper-Rayleigh scattering technique. The method is free from the overestimation which is encountered in the conventional method due to the fluorescence induced by two-photon absorption.

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.

The title compound (I) shows a 21-fold two-photon excited fluorescence enhancement in response to H2S.

Reactions of dl - and meso -stilbene dibromides with 2-nitropropan-2-yl anion in DMSO proceed by the E2 mechanism, whereas that of erythro - p -nitrostilbene dibromide proceeds by a competing ionic and SET mechanism.

Elimination reactions of (E)-O-p-nitrophenyl-2,4-dinitrobenzaldoxime promoted by triethylamine in MeCNH2O proceeded by the E2 mechanism. The rate decreased as the mole % of MeCN increased up to 70 % and then increased upon further increase in the MeCN concentration. The results have been interpreted with the solvent effect.

We report a two-photon fluorescent probe (SHP-Mito) which can ratiometrically detect mitochondrial H(2)O(2) in live cells and intact tissues at100 μm depth through the use of two-photon microscopy.

We report a two-photon turn-on probe (AS1) that can be excited by 780 nm femto-second pulses and visualize glucose uptake and the changes in the intracellular glucose concentration in live cells and tissue by two-photon microscopy.

Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.

Molecular imaging by two-photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more-versatile tool, a large variety of two-photon probes are needed. Herein, we report a new two-photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni(2+) ions in fresh fish organs at 90-175 μm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni(2+) ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two-photon-excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP-MS), thereby allowing the quantitative measurement of Ni(2+) ions in live tissue.

We report a two-photon fluorescent probe (ACu1) that can be excited by 750 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize Cu(+) distribution in live cells and tissues by two-photon microscopy.

We report a two-photon probe (SZn-Mito) for mitochondrial zinc ions ([Zn2+]m). This probe shows a 7-fold enhancement of two-photon-excited fluorescence in response to Zn2+ with a dissociation constant (Kd(TP)) of 3.1 ± 0.1 nM and pH insensitivity in the biologically relevant range, allowing the detection of [Zn2+]m in a rat hippocampal slice at a depth of 100−200 μm without interference from other metal ions through the use of two-photon microscopy.

Two-photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two-photon probes for specific applications are needed. In this context, many research groups are developing two-photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two-photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two-photon probes for various in vivo imaging applications.

A naphthalimide-based highly selective colorimetric and ratiometric fluorescent probe for the fluoride ion displayed both one- and two-photon ratiometric changes. Upon reaction with the F(-) (TBA(+) and Na(+) salts) anion in CH(3)CN as well as in aqueous buffer solution, probe 1 shows dramatic color changes from colorless to jade-green and remarkable ratiometric fluorescence enhancements signals. These properties are mechanistically ascribed to a fluoride-triggered Si-O bond cleavage that resulted in a green fluorescent 4-amino-1,8-naphthalimide.