Synthesis and characterization of water-soluble two-photon excited blue fluorescent chromophores for bioimaging.

We report here the synthesis and characterization of a new type of non-ionic blue fluorescent water-soluble chromophores specifically designed for two-photon absorption microscopy. The water solubility is induced by introduction of short oligo(ethylene glycol) monomethyl ether moieties. This work has led to low molecular weight dyes with efficient two-photon absorption cross sections and high fluorescence quantum yield in organic solvents as well as in aqueous solutions.     

Multicolor two-photon tissue imaging by wavelength mixing

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.     

The nonlinear effect of the composite influence of red and blue light on <Emphasis Type="Italic">Escherichia coli</Emphasis> viability

A nonlinear dependence of E. coli cell inhibition was found during the simultaneous irradiation by the blue and red spectral regions at a power density of 100 mW/cm². This dependence can be explained by the cascade two-photon light absorption by DNA molecules with intermediate resonance at the cellular chromophores, which leads to excitation and subsequent DNA damage similar to the damage that is caused by exposure to UV quanta.     

An amphiphilic ruthenium(II)-polypyridyl appended porphyrin as potential bifunctional two-photon tumor-imaging and photodynamic therapeutic agent

An amphiphilic porphyrin appended with a Ru(II)-polypyridyl complex (Ru-P) showing a moderate two-photon absorption cross-section (178.0 ± 26.8 GM), high singlet oxygen quantum yield and rapid cellular uptake was synthesized. In vitro study using human nasopharyngeal carcinoma cells showed that Ru-P exhibited a strong two-photon induced fluorescence upon uptake, lysosomal localization and potent two-photon induced cytotoxicity. These results show that Ru-P, which was designed to enhance its cellular uptake, can potentially be used as an efficacious bifunctional two-photon tumor-imaging and photodynamic therapeutic agent despite its moderate two-photon absorption cross-section.     

Photochemical revelation of the stacking aggregation of thymine chromophores in water solutions of polythymidylic acid

The structure heterogeneity of water solutions of polyribothymidylic acid at T(room) was studied from changes caused in their absorption spectra by the photodimerization reaction. Three fractions of thymine chromophores were revealed from the differential absorption spectra: (a) the main fraction consisting of weakly interacting (isolated chromophores) chromophores with the absorption spectrum maximum at approximately 270 nm; (b) pair chromophores of the first type with the absorption spectrum maxima at 260 and 290 nm (exciton splitting 4000 cm(-1)); and (c) pair chromophores of the second type with the absorption spectrum maxima at 250 and 280 nm (exciton splitting 4300 cm(-1)). The revealed aggregates have a relatively high photochemical activity in the photodimerization reaction in comparison with the isolated chromophores. They contribute little to the total absorption spectrum of solutions but make a great contribution to its changes at the initial stages of the UV irradiation of solutions.     

N-phenyl-carbazole-based two-photon fluorescent probes: strong sequence dependence of the duplex vs quadruplex selectivity

Herein we report on the synthesis and DNA recognition properties of a series of three N-phenyl carbazole-based light-up probes initially designed for two-photon absorption. The vinylic derivatives (Cbz-2Py, Cbz-3Py) display strong fluorescence enhancement when bound to various duplex- and quadruplex-forming oligonucleotides whereas the oxazole derivative is not fluorescent in DNA. Determination of affinity constants by fluorimetric titrations evidenced that Cbz-2Py has a clear preference for AT-rich duplex structures. Circular Dichroism (CD) measurements confirmed the sequence-dependent binding of this compound and suggest insertion in the minor groove as shown by a strong induced CD (ICD) signal and further supported by molecular modeling. Altogether the data indicate that duplex vs quadruplex selectivity of the dyes is strongly dependent on the sequence of the duplex. Finally, the dyes exhibit high two-photon absorption cross-sections (up to 540 GM in glycerol) and allow a fine and bright staining of nuclear DNA with low background fluorescence as shown by one and two-photon confocal microscopy imaging of fixed cells.     

Molecular modeling of two-photon absorption and third-order nonlinearities of polymethine dyes for all-optical switching

Stimulated by a recent experimental report [Hales JM et al. (2010) Science 327:1485-1488], two-photon absorption and third-order optical nonlinearities of selenopyrylium- and bis(dioxaborine)-terminated polymethine dyes (called SE-7C and DOB-9C) used for all-optical switching were investigated theoretically with time-dependent DFT (TD-DFT) and response theory as well as visualized real-space analysis. The calculated results for the first hyperpolarizability and second hyperpolarizability demonstrated that the two molecules both have large third-order optical nonlinearities. Using real-space analysis, we were able to visually determine that in the one-photon absorption (OPA) process, the first singlet excited state of SE-7C and DOB-9C is an intramolecular charge transfer (ICT) excited state with strong absorption, while the second excited state of these dyes (also termed the "ICT state") shows weak absorption. However, in the two-photon absorption (TPA) process, a larger TPA absorption cross-section was predicted for the second excited state. In this paper, we describe the properties of the S2 excited state, incorporating charge transfer and the transition moment, via real-space analysis, which was very important for understanding the TPA characteristics of the S(2) state.     

生物分子的双光子激发发光

应用Nd:YAG激光器和光学光谱分析仪对蛋白质分子的同时吸收双光子过程作了进一步研究,讨论了蛋白质分子双光子过程的特性和估计了它们的双光子吸收截面,并得到胰蛋白酶,白蛋白和色氨酸等分子由于双光子激发产生的荧光光谱.     

Molecular docking of novel donor–π–acceptor dicyanomethylenedihydrofuran-based fluorescent chromophores

A series of donor–π–acceptor dicyanomethylenedihydrofuran (DCDHF)-based chromophores comprising different π-aryl bridges and different terminal groups was synthesized and characterized. The chromophores were synthesized via Knoevenagel condensation of the active methyl-bearing DCDHF (electron acceptor) with a tertiary amine-containing arylaldehyde (electron donor) in dry pyridine at room temperature in the presence of a few drops of acetic acid. The synthesis approach involved the development of phenyl(thienyl)vinyl-bridged dicyanomethylenedihydrofuran dyes with a tertiary amine terminal group. Both absorption and emission spectra were explored. The strong emission properties detected using the synthesized chromophores could be attributed to intramolecular charge transfer. The chemical structures of the synthesized chromophores were verified using ¹H/¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopy. Both tertiary amine-containing and arylaldehyde groups were found to influence the biological properties of the synthesized chromophores. The synthesized (DCDHF)-based hybrids were tested to examine antibacterial effectiveness. Derivatives 1 and 2 demonstrated activity towards Gram-positive bacteria rather than Gram-negative bacteria when compared with an amoxicillin antibiotic reference. Finally, molecular docking inspiration was undertaken to determine their binding relationships (PDB code: 1LNZ).     

Intermolecular interaction of nickel (ii) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin: A multi-technique study

The interaction of nickel (II) phthalocyanine tetrasulfonic acid tetrasodium salt with bovine serum albumin (BSA) has been investigated by combination of fluorescence, UV-vis absorption, Fourier transform infrared (FT-IR), and circular dichorism (CD) spectroscopies as well as through molecular docking. Fluorescence quenching and absorption spectra were investigated as a mean for estimating the binding parameters. Analysis of fluorescence quenching data at different temperatures was performed in order to specify the thermodynamics parameters for interactions of phthalocyanine complex with BSA. According to experimental data it was suggested that phthalocyanine had a significant binding affinity to BSA and the process was entropy driven. Based on the results of molecular docking it was indicated that the main active binding site for this phthalocyanine complex is site I in subdomain IIA of BSA. The results provide useful information for understanding the binding mechanism of anticancer drug-albumin and gives insight into the biological activity and metabolism of the drug in blood.     

Zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine as a new site-specific marker for serum albumin

The interaction of zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine with bovine serum albumin was studied via electron absorption spectroscopy and fluorescence spectroscopy. Being the associate in aqueous media, zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine is converted to its monomeric form to be bound to albumin, with the monomeric form of the phthalocyanine being characterized by a fluorescence in the visible region of the spectrum. The displacement titration of albumin complexes with the known site-specific markers (such as warfarin, L-tryptophan, and hemin) and phthalocyanine was performed to show the location of zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine within the IIA subdomain of albumin.     

Correction of spectra for studying dye aggregates by resonance light scattering

Resonance light scattering (RLS), a phenomenon of abrupt enhancement of Rayleigh light scattering in close proximity to an absorption band, is easily detectable in solutions of strongly absorbing chromophores, which form large aggregates with strong π-electronic coupling among the chromophores. RLS spectra need to be corrected for the sensitivity of the spectrofluorimeter as well as for the effects of internal light filter. A method for correcting the measured RLS is described. It was shown by the method that addition of KCl induces formation of extended supramolecular aggregates (probably of H-type) of the anionic dye merocyanine 540 in water. The RLS spectra of a photosensitizer m-tetra(hydroxyphenyl)chlorin (Foscan®) indicate formation of J-aggregates of this dye in aqueous medium.     

Coumarin derived chromophores in the donor-acceptor-donor format that gives fluorescence enhancement and large two-photon activity in presence of specific metal ions

Two coumarin derived dyes (compounds L_a and L) have been synthesized in high yields by Schiff base condensation. On probing with a femtosecond laser, none of these compounds show any two-photon activity in the wavelength range, 760-860 nm. However, L_a in presence of Zn(II) and L in presence of Mg(II), exhibit large two-photon absorption as well as emission in the same wavelength range. Theoretical calculations at the B3LYP functional with 6-31G∗ and LanL2DZ mixed basis set under DFT formalism support experimental results.     

Major signal increase in fluorescence microscopy through dark-state relaxation

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.     

Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.     

别藻蓝蛋白藻蓝胆素发色团分子构象研究

主要研究了蓝绿藻污棕席藻(Phormidium luridum)别藻蓝蛋白在不同 pH值条件下的吸收光谱和共振拉曼光谱.发现低聚化的结果导致了三聚体别藻蓝蛋白 650nm 特征吸收峰的消失和一些共振拉曼带强度和位置的移动.结果表明在低 pH 值作用下的低聚化的别藻蓝蛋白中藻蓝胆素发色团分子的构象和自由胆素分子类似,比三聚体的别藻蓝蛋白的发色团分子更趋于卷曲,折叠的构象态.而三聚体的别藻蓝蛋白,主要的拉曼带 1645cm^-1是其发色团分子构象处于更线性延展的标志,其光谱行为和吸收光谱 A_vis/A_uv所表征的发色团分子构象的结果相一致.     

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.     

The efficiency of two-photon photolysis of a "caged" fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals

Localized photolysis of caged neurotransmitters with the two-photon effect for investigations at synaptic preparations was evaluated by determining the toxicity to synaptic transmission of pulsed near-IR laser light focused into the terminals of the snake neuromuscular junction, and measuring the extent of photolysis of a conventional caging group with similar irradiation in microcuvette experiments. Photodamage was seen in synaptic terminals as a large, irreversible increase of spontaneous synaptic activity with laser flashes of 5 ms at 1 Hz at average powers > 5 mW and was due to multiphoton absorption. Localized photolysis due to two-photon absorption was investigated for a representative caged fluorophore, the 1-(2-nitrophenyl)ethyl ether of pyranine (NPE-HPTS). Irradiation of NPE-HPTS at 5 mW with the same optical arrangement produced very low rates of photolysis. NPE-HPTS photolysis mechanisms were investigated at high laser powers by measuring (1) the kinetics of two-photon fluorescence generated by two-photon photolysis in the focal volume and (2) the rates of HPTS accumulation inside closed 2-10 microm radius vesicles, measured with one-photon excitation during two-photon photolysis by repetitive 10 micros laser exposures. The two-photon crosssection of NPE-HPTS photolysis calculated from the rates is 0.02-0.04 GM (10(-50) cm4 x s/photon) and limits the efficiency of photolysis at 5 mW. With free diffusional exchange, 50% steady-state cage depletion in the focal volume was estimated to occur only at high laser powers of ca. 72 mW, masked in experiments by multiphoton bleaching. Based on these results, the two-photon photolysis cross-section needed for 50% steady-state photolysis of a caged neurotransmitter at 5 mW is calculated as 31 GM, much higher than in existing caged compounds.     

The donor-acceptor biphenyl platform: a versatile chromophore for the engineering of highly efficient two-photon sensitive photoremovable protecting groups

Different photoremovable protecting groups in the o-nitrobenzyl, phenacyl, and 2-(o-nitrophenyl)propyl series with a donor-acceptor biphenyl backbone, known to display excellent two-photon absorption cross-sections, were investigated in order to develop efficient two-photon sensitive photoremovable protecting groups. The 2-(o-nitrophenyl)propyl series was a more versatile platform to increase the two-photon sensitivity of photoremovable protecting groups, leading to the p-alkoxy and p-bisalkylamino-4-nitro-[1,1'-biphenyl]-3-yl)propyl derivatives: PENB and EANBP respectively. Those two photoremovable protecting groups are to date the best caging groups for two-photon excitation at 800 and 740 nm respectively, offering attracting perspectives in chemical biology.