Two-photon Photophysical Properties Research Articles

β-β-alkynyl-bridged BODIPY dimers: Synthesis, one- and two-photon photophysical properties

Six β-β-linked BODIPY dimers with different EDG-EDG and EDG-EWG were synthesized by changing the nature of the EDG and EWG at the meso position. The fluorescence lifetimes and quantum yield of z4 and z5 with the same substituent groups at the two meso positions are much higher than those of the BODIPY dimers with two different substituents. Compound z4 exhibits the highest fluorescence quantum yield (11.24 %) and fluorescence lifetime (1.32 ns). Both compounds z4 and z5 show important two-photon induced fluorescence in the 800–1000 nm region with a clear quadratic dependency of the emission intensity vs the laser excitation power.

Push-pull conjugated chromene-derivatives for potential bio-imaging applications. Synthesis, X-Ray and DFT studies, one- and two-photon photophysical properties

Dicyanomethylene-4H-benzopyran derivatives dyes containing D-π-A-type structure have been prepared and fully characterized. The Z configuration of all three structures has been confirmed by SCXRD studies. Dyes 1–6 present a strong absorption in the 450–550 nm region with ε values over 43 000 M−1 cm−11–4 showed fluorescence properties while 5 and 6 are very weakly fluorescent. The electronic structure of the first singlet excited state S1 was investigated to further explain the evolution of fluorescence quantum yields. The two-photon excitation spectra were recorded with a similar behaviour upon two-photon excitation, with two absorption bands, one in the 800–850 nm region with σ2 of 200–300 GM and another one at about 1000 nm with σ2 of 300–400 GM. Dyes 3 and 4 have shown interesting properties (both in intensity and FLIM modes) in two-photon excited microscopy.

Polyanionic Polydentate Europium Complexes as Ultrabright One- or Two-photon Bioprobes.

A family of europium (III) complexes based on a polydentate ligand functionalized by charge-transfer antennae presents remarkable one- and two-photon photophysical proper-ties in water or buffer. A detailed analysis of their emission properties suggests that the wrapping of the ligand around the central rare-earth ion results in an overall Cs symmetry in agreement with the theoretical simulation and that about 65-70 % of the emission intensity is concentrated in the hypersensitive 5 D0 →7 F2 transition at 615 nm. Their brightness is excellent, in the range of the best lanthanide bioprobes making them very attractive for bio-imaging experiments.

Two-Photon Absorption Properties and Structures of BODIPY and Its Dyad, Triad and Tetrad.

A series consisting of a dyad, a triad and a tetrad containing either two, three and four BODIPY units, respectively, has been synthesized and fully characterized and compared to two mono-BODIPY analogs (used as references). The one- and two-photon photophysical properties have been measured and the X-ray structures of four of the BODIPY derivatives have been determined. In the 700-900 nm range, the two-photon absorption (TPA) cross sections range from 30 GM to 160 GM for these compounds.

Amino-Si-rhodamines: A new class of two-photon fluorescent dyes with intrinsic targeting ability for lysosomes

Noninvasive and specific visualization of lysosomes by fluorescence technology is critical for studying lysosomal trafficking in health and disease and for evaluating new cancer therapeutics that target tumor cell lysosomes. To date, there are two basic types of lysosomal probes whose lysosomal localization correlates with lysosomal acidity and endocytosis pathway, respectively. However, the former may suffer from pH-sensitive lysosomal localization and alkalization-induced lysosomal enzyme inactivation, and the latter need long incubation time to penetrate cell membrane due to the energy-dependency of endocytosis process. In this work, a new class of two-photon fluorescent dyes, termed amino-Si-rhodamines (ASiRs), were developed, which possess the intrinsic lysosome-targeted ability that is independent of lysosomal acidity and endocytosis pathway. As a result, ASiRs show not only the stable lysosomal localization against lysosomal pH changes and negligible interference to lysosomal function, but also excellent cell-membrane-permeability due to the energy-independent passive diffusion pathway. These merits, coupled with their excellent two-photon photophysical properties, long-term retention ability in lysosomes, and negligible cytotoxicity, make ASiRs very suitable for real-time and long-term tracking of lysosomes in living cells or tissues without interference to normal cellular processes. Moreover, the easy functionalization via amino linker further allows the construction of various fluorescent probes for biological targets of interest based on ASiR skeleton, as indicated by the cancer-targeted fluorescent probe ASiR6 as well as a fluorescent peroxynitrite probe ASiR-P.

π-extended porphyrin dimers as efficient near-infrared emitters and two-photon absorbers

A Zn-porphyrin dimer and its linear conjugate with two diketopyrrolopyrrole (DPP) units have been prepared and their one- and two-photon photophysical properties have been characterised. The already wide conjugation of the dimer is further extended in the DPP containing derivative, leading to absorption and emission features in the near-infrared. The fluorescence quantum yield of the two compounds is of the order of 0.20 in organic solvents, a remarkable value for NIR emitting Zn-porphyrin systems. High two-photon absorption cross sections of 10,000 GM and 21,500 GM are measured at 910 nm for the dimer and the DPP conjugate, respectively, making these dimers promising for two-photon excited fluorescence imaging applications.

Photostability Enhancement of Fluorenone-Based Two-Photon Fluorescent Probes for Cellular Nucleus Monitoring and Imaging

A series of fluorenone-based two-photon fluorescent probes with high photostability for nucleus imaging are prepared and developed. The one- and two-photon photophysical properties exhibit these new probes possess 0.448–0.634 of fluorescence quantum yields and 469–495 GM of two-photon absorption cross sections at 800 nm femtosecond laser excitation. The luminescence “turn-on” experiment in buffer solutions indicates that 35-fold of fluorescence intensity and 68-fold fluorescence quantum yield enhancement appear between new probes and calf thymus DNA. In the nuclear double-staining experiment, the high mean colocalization coefficients of 0.92–0.96 between new probes and nuclear labeling dye Hoechst 33342 are acquired, demonstrating excellent nuclear localization in 3T3 cells. The counterstain studies by introducing commercial mitochondrial staining reagent MTR and nuclear staining dye DAPI further show good membrane permeability and counterstain compatibility in multicolor cell labeling application. The ph...

An Amphiphilic BODIPY-Porphyrin Conjugate: Intense Two-Photon Absorption and Rapid Cellular Uptake for Two-Photon-Induced Imaging and Photodynamic Therapy.

The new amphiphilic BODPY-porphyrin conjugate BZnPP and its precursor BZnPH were synthesised, and their linear and two-photon photophysical properties, together with their cellular uptake and photo-cytotoxicity, were studied. This amphiphilic conjugate consists of a hydrophobic BODIPY moiety and a hydrophilic tetra(ethylene glycol) chain bridging a cationic triphenylphosphonium group to an amphiphilic porphyrin ZnP through acetylide linkers at its meso positions. A large two-photon absorption cross-section (σ=1725 GM) and a high singlet oxygen quantum yield (0.52) were recorded. Intense linear- and two-photon-induced red emissions were also observed for both BZnPP and BZnPH. Further in vitro studies showed that BZnPP exhibited very efficient cellular uptake and strong photocytotoxic but weak dark cytotoxic properties towards human breast carcinoma MCF-7 cells. In summary, the two-photon-induced emission and the potent photo-cytotoxicity of BZnPP make it an efficacious dual-purpose tumour-imaging and photodynamic therapeutic agent in the tissue-transparent spectral windows.

Polymer Monoliths Containing Two-Photon Absorbing Phenylenevinylene Platinum(II) Acetylide Chromophores for Optical Power Limiting.

A series of platinum(II) acetylide complexes containing p-phenylenevinylene and moieties end-capped with triphenylamine groups have been incorporated into poly(methyl methacrylate) (PMMA) monoliths for optical power limiting applications. The one- and two-photon photophysical properties were investigated and compared to the photophysical properties in THF. The absolute two-photon absorption cross-section values for the monolith samples were measured and are comparable to the values obtained in solution. In the PMMA monoliths, the complexes retained the important two-photon absorption and reverse saturable absorption properties necessary for optical power limiting via dual mode mechanism, and their strong nonlinear absorption property was demonstrated by the open-aperture Z-scan method. Photostability studies of the p-phenylenevinylene platinum(II) acetylide complexes showed two photodegradation processes: a trans-to-cis isomerization and a singlet-oxygen sensitized self-oxidative cleavage. The photostability of the least photostable complex TPV0 was increased upon incorporation into a PMMA matrix.

A specific nucleophilic ring-opening reaction of aziridines as a unique platform for the construction of hydrogen polysulfides sensors.

A hydrogen polysulfide mediated aziridine ring-opening reaction was discovered. Based on this reaction, a novel H2Sn-specific chemosensor (AP) was developed. AP showed high sensitivity and selectivity for H2Sn. Notably, the fluorescent turn-on product (1) exhibited excellent two-photon photophysical properties, a large Stokes shift, and high solid state luminescent efficiency.

Facile synthesis, optical properties, and live cells imaging of two novel hydrophilic two-photon fluorophores with long-wavelength emission

Two novel organic compounds, TPIM1 and TPIM2, with hydroxyl terminal hydrophilicity groups, have been designed and synthesized. Their one- and two-photon photophysical properties have been experimentally investigated, and emission wavelengths are 591 and 595 nm in phosphate buffered saline (PBS), respectively. The two-photon action cross-sections of two compounds were estimated by two-photon excited fluorescence technique using 200 fs, 76 MHz, Ti-sapphire laser, which are 95 and 70 GM for TPIM1 and TPIM2 in PBS, respectively. The photostability and cytotoxicity were evaluated to demonstrate the potential utility of TPIM1 as a suitable standalone fluorophore for live cells two-photon fluorescence biological imaging. TPFM imaging results demonstrate that HepG2 treated with 10 μM TPIM1 provides bright images with negligible toxicity.

High Efficiency Platinum Acetylide Nonlinear Absorption Chromophores Covalently Linked to Poly(methyl methacrylate)

We report three platinum acetylide acrylate monomers containing known two-photon absorption (TPA) chromophores and their covalent incorporation into polymers via free radical polymerization with methyl methacrylate. The photophysical properties of the platinum acetylide monomers and resulting poly(methyl methacrylate) (PMMA) copolymers were investigated to determine if the one- and two-photon photophysical properties of the chromophores were maintained in the copolymers. The photophysical properties of the series of copolymers were studied in solution and solid state with minimum shifts exhibited in the ground state absorption, photoluminescence, and triplet-triplet transient absorption spectra. The polymer films displayed markedly stronger phosphorescence and longer triplet excited state lifetimes than the polymers in solution or the monomers. The incorporation of the platinum acetylide chromophores into the PMMA copolymers allows the materials to be cast as thin films or into free-standing monoliths. Films with ~3.6 μm in thickness and monoliths with 1 mm path length were fabricated and examined. The nonlinear absorption responses of the polymers in solution were measured via the nanosecond z-scan method, and the solid state polymer monoliths were measured via nonlinear transmittance. Both measurements indicate that the polymers exhibited strong transmittance attenuation at input pulse energies exceeding 100 μJ.

Enhanced Singlet Oxygen Generation from a Porphyrin–Rhodamine B Dyad by Two‐Photon Excitation through Resonance Energy Transfer

Mitochondrial-targeting photosensitizers have been associated with effective photodynamic responses. However, most photosensitizers absorb light between 400 and 700 nm, where light penetration through tissues is limited. Two-photon excitation is a rational approach to improve light penetration through tissues. In this report, the two-photon photophysical properties of a porphyrin-rhodamine B conjugate (TPP-Rh), previously demonstrated to target the mitochondria, were evaluated. The properties studied included: two-photon absorption (TPA) cross sections (σ2 ); resonance energy transfer (RET) kinetics and dynamics; and singlet oxygen generation. The conjugation of Rh B to TPP-OH approximately doubled the σ2 of TPP-Rh at 800 nm (40 ± 4 GM) compared with the parent porphyrin, TPP-OH (16 ± 4 GM). Furthermore, the rate of DPBF oxidation by singlet oxygen generated from TPP-Rh was twice as fast compared with that from TPP-OH (73 % versus 33% in 10 min) following two-photon excitation at 800 nm. In addition, a significantly stronger luminescence signal was detected from TPP-Rh, than from TPP-OH at 1270 nm, following two-photon excitation. This study indicates that conjugating photosensitizers to Rh B could provide greater TPA at the near-infrared range in addition to preferential mitochondrial accumulation for improved photodynamic responses.

Significant effects of branches and bromine substitution of near-infrared two-photon photosensitizers on the generation of singlet oxygen

This letter reports the influence of branches and bromine substitution of the photosensitizers on one- and two-photon absorption generation of singlet oxygen. Near-infrared femtosecond Ti:squassier laser was utilized to determine two-photon properties of the photosensitizers tuning wavelength from 700 to 880 nm at intervals of 20 nm. One- and two-photon optical and photophysical properties of the photosensitizers show significant dependence on the branches and substituted bromine atoms.

Highly Selective Mitochondria-Targeting Amphiphilic Silicon(IV) Phthalocyanines with Axially Ligated Rhodamine B for Photodynamic Therapy

Two axially ligated rhodamine-Si(IV)-phthalocyanine (Rh-SiPc) conjugates, bearing one and two rhodamine B, were synthesized and their linear and two-photon photophysical, subcellular localization and photocytotoxic properties were studied. These Rh-SiPc conjugates exhibited an almost exclusive mitochondrial localizing property in human nasopharyngeal carcinoma (HK-1) cells and human cervical carcinoma (HeLa) cells. Strong photocytotoxic but low dark cytotoxic properties were also observed for the two Rh-SiPc conjugates toward the HK-1 cells. Using nuclei staining method and flow cytometric DNA content analysis, apoptotic cell death was induced by these conjugates upon photoactivation. This observation is consistent with their mitochondrial localization property. The observed properties of these conjugates qualify them as promising PDT agents.

Boron-Containing Two-Photon-Absorbing Chromophores. 3. One- and Two-Photon Photophysical Properties of p-Carborane-Containing Fluorescent Bioprobes

Boron-containing two-photon-absorbing fluorophores have been prepared as new bifunctional molecules, potentially useful in two-photon excited microscopy (TPEM) and boron neutron capture therapy. They are based on a one-dimensional conjugated system containing a p-carborane entity at one end of the molecule and various electron-donating groups containing oxygen or nitrogen atoms at the other end. We investigated their one- and two-photon photophysical properties. They showed efficient fluorescence in an organic solvent, as well as in water for two of them, allowing microscopy on cell cultures. High two-photon absorption cross sections were determined in the 700-900 nm range. TPEM images were obtained with these new p-carborane-containing fluorophores, with laser intensities in the submilliwatt range.

Abstract 497: Mitochondria-targeting photosensitizers activable by two-photon absorption

Abstract Two-photon absorption (TPA) is a non-linear optical process, used to activate photosensitizers in the NIR region (700 - 850 nm) thus affording greater tissue penetration in photodynamic therapy (PDT). Since the efficiency of this process has a quadratic dependence on the light intensity, the photodynamic effects are restricted to a small area around the laser's focal point, thus providing spatial selectivity. Although most clinically approved photosensitizers can be activated by TPA, they generally have very small cross-sections (generally below 10 GM around 800 nm). Thus, light intensities required to activate them could damage normal surrounding tissues. The TPA of photosensitizers can however be improved, without perturbing their ability to generate singlet oxygen, by conjugating them to effective TPA chromophores. These then serve as TPA antennae and upon activation transfer their energy to the photosensitizer which then generates singlet oxygen for PDT. In this study a porphyrin-rhodamine B dyad (TPP-Rh), previously demonstrated to target mitochondria, was evaluated as a two-photon photosensitizer for PDT. TPP-Rh, was synthesized by conjugating a monohydroxy porphyrin photosensitizer (TPP-OH) to rhodamine B (Rh B), an effective TPA chromophore, via a saturated hydrocarbon linker, thus enhancing intramolecular FRET between the dyad components and ultimately the photosensitizer's TPA. To evaluate the efficiency of TPP-Rh as a two-photon photosensitizer we studied both its one and two-photon absorption photophysical properties in comparison to those of its individual components: Rh B and TPP-OH. These included: FRET kinetics, FRET dynamics, and the singlet oxygen generation kinetics. A FRET rate of 1.679 × 1012 s−1 and a 99 % FRET efficiency was calculated for TPP-Rh from its spectral properties suggesting efficient FRET from the TPA moiety (Rh B) to the photosensitizer moiety (TPP). In addition to this, TPP-Rh produced singlet oxygen at comparable rates when excited with both TPA at 800 nm and one photon absorption (OPA) at 650 nm. Thus, this indicates that conjugating photosensitizers to rhodamine B could provide several advantages in PDT: deeper tissue penetration, spatial selectivity and selective photosensitizer accumulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 497.

Effect of Bridging Position on the Two-Photon Polymerization Initiating Efficiencies of Novel Coumarin/Benzylidene Cyclopentanone Dyes

One- and two-photon photophysical and photochemical properties of dyes 3-DAC and 4-DAC containing coumarin and benzylidene cyclopentanone moieties were studied. Their ground state configurations were optimized using both Hartree-Fock and density functional theory (B3LYP functional) methods, and excited-state properties were calculated using time-dependent density functional theory. These two dyes share the same formula and possess similar structures, except for different bridging positions between the benzylidene cyclopentanone and coumarin moieties. The bridging position was found to have a significant effect on the electronic structure and photophysical and photochemical properties of the dyes. 3-DAC shows higher conjugation and is more planar than 4-DAC, and it exhibits a larger TPA cross section. In contrast, 4-DAC has a twisted conformation, exhibits a lower electron transfer free energy with initiator and shows higher sensitizing efficiencies in one-photon polymerization. Furthermore, the application potential of 3-DAC and 4-DAC in two-photon polymerization (TPP) was studied. Both dyes could be used directly as initiators in TPP. The TPP threshold energies of the corresponding resins were as low as the best reported results. High-resolution 2D and 3D nanopatterns containing low amounts of small molecule residue were successfully fabricated by TPP, demonstrating the extensive application prospects of these dyes in the fabrication of micromachines, microsensor arrays and biomedical devices.

TPA-active D–π–D fluorophores with rigid, planar cores from phenylene to indenofluorene and indolocarbazole

A series of D–π–D quadrupolar fluorophores with systematically varied π-conjugated cores were prepared using the Heck reaction and their single- and two-photon photophysical properties were examined. Introduction of extended π-conjugated cores such as indenofluorene/indolocarbazole was found to induce only small shifts in the absorption and emission maxima versus the phenylene linked fluorophore but greatly enhance their single- and two-photon absorption cross-sections. The effects of positional substitution on the properties of the indolocarbazole derivatives were also investigated.

Boron Containing Two-Photon Absorbing Chromophores. 2. Fine Tuning of the One- and Two-Photon Photophysical Properties of Pyrazabole Based Fluorescent Bioprobes

New boron containing two-photon absorbing fluorophores have been prepared. Centered on a pyrazabole central core, various conjugated systems and end groups were investigated to modulate their physicochemical properties (alkoxy, diphenylamino, and boron dipyromethene groups). One and two-photon photophysical characterizations were performed, showing efficient fluorescence in organic solvents. High two-photon absorption cross sections were determined in the 500-800 nm range. Two-photon excited microscopy images were also obtained with these new boron containing fluorescent bioprobes with laser intensities in the milliwatt range.