Cellular Detection of a Mitochondria Targeted Brominated Vinyl Triphenylamine Optical Probe (TP−Br) by X-Ray Fluorescence Microscopy

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP−2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP−2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP−RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP−Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP−Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP−Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.     

Effect of Substitution Site on Two-Photon Absorption and Excited States Properties of Lipid Droplets Detection NAPBr Dyes: a Theoretical Perspective

Two-photon fluorescence dyes have shown promising applications in biomedical imaging. However, the substitution site effect on geometric structures and photophysical properties of fluorescence dyes is rarely illustrated in detail. In this work, a series of new lipid droplets detection dyes are designed and studied, molecular optical properties and non-radiative transitions are analyzed. The intramolecular weak interaction and electron-hole analysis reveal its inner mechanisms. All dyes are proven to possess excellent photophysical properties with high fluorescence quantum efficiency and large stokes shift as well as remarkable two-photon absorption cross section. Our work reasonably elucidates the experimental measurements and the effects of substitution site on two-photon absorption and excited states properties of lipid droplets detection NAPBr dyes are highlighted, which could provide a theoretical perspective for designing efficient organic dyes for lipid droplets detection in biology and medicine fields.     

Syntheses of Novel Asymmetric Cyclopentanone Dyes and Measurement of Two－photon Absorption Cross－section

A simple synthesis route with a high yield of novel asymmetric cyclopentanone dyes 3a—3e and their highly two-photon up-converted fluorescences are reported. The dyes have good solubilities in most of ordi-nary solvents, a wide UV absorption wavelength range from 380—540 nm, and high fluorescence quantum yields. The two-photon absorption cross-sections of dyes 3a—M were measured in chloroform by a two-pho-ton induced fluorescence method. All of these properties of the new dyes make them suitable for being used as two-photon fluorescent probes.     

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe ( DAOTA-M2 ) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.     

Conformationally restricted dipyrromethene boron difluoride (BODIPY) dyes: highly fluorescent, multicolored probes for cellular imaging

Novel fluorescent, conformationally restricted dipyrromethene boron difluoride (BODIPY) dyes have been prepared by introducing a naphthalenyl group at the meso position of the BODIPY core. These BODIPY dyes exhibit increased fluorescence quantum yields compared with dyes that have a meso-position phenyl group with internal rotation. The absorption and emission wavelengths of such conformationally restricted BODIPY dyes can be easily tuned to the near-IR range by derivatization through a condensation reaction with benzaldehyde derivatives. The two-photon absorption properties of these BODIPY dyes were also investigated and the results show that they exhibit increased two-photon excited fluorescence compared to analogue dyes that contain a phenyl group. The one- and two-photon fluorescence imaging of living cells by using selected BODIPY dyes has been successfully demonstrated.     

金属配合物在双光子荧光探针中的应用研究

金属配合物因其优异的光物理性质,如配位结构可调、好的光稳定性、大的斯托克位移、高的荧光量子产率与长的荧光寿命等,在生物成像、分子探针、医学影像等领域中备受关注.与单光子吸收相比,双光子吸收的金属配合物因其具有更加优秀的深度分辨率以及低光损伤性等优点,近些年被广泛应用于生物分子的荧光探针和细胞器染料等.本文综述了近年来具...     

Interaction of Crown Ether‐Annelated Styryl Dyes with Double‐Stranded DNA

DNA‐binding properties of 15‐crown‐5‐derived mono‐ and bis‐styryl dyes were investigated in the presence of calf thymus DNA. To access the factors that influence the DNA association in the series of these ligands, the structure of the molecules was varied by either changing size of the heterocyclic moiety or altering the position of the styryl substituents. The major binding mode for the monostyryl dyes is intercalation. Notably, binding of the dyes to the nucleic acids leads to a fluorescence enhancement by a factor of up to 54. Therefore, these cationic styryl derivatives may be applied as fluorescent “light‐up” probes for DNA detection.     

Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.     

Polyfluorene based conjugated polymer nanoparticles for two-photon live cell imaging

Two-photon excitation microscopy (2PEM) has been known as a noninvasive and powerful bio-imaging tool for studying living cells, intact tissues and living animals because of their unique advantages such as localized excitation, deep tissue penetration as well as less photo-damage. However, the major limitations that hinder its practical applications in biological systems are low two-photon absorption cross sections of conventional fluorescence probes. Conjugated polymer nanoparticles (CPNs) consisting of highly fluorescent conjugated polymers are promising fluorescent probes for 2PEM due to their unique advantages including large two-photon absorption cross sections, high fluorescence quantum yield, good photo-stability and biocompatibility, facile chemical synthesis, tunable optical properties as well as versatile surface modifications. This account summarizes the recent efforts of our group on development of novel polyfluorene based CPNs as 2PEM contrast agents for live cell imaging.     

Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.     

Novel 2,1,3-benzothiadiazole-based red-fluorescent dyes with enhanced two-photon absorption cross-sections

This paper reports the two-photon absorbing and orange-red fluorescence emitting properties of a series of new 2,1,3-benzothiadiazole (BTD)-based D-pi-A-pi-D-type and star-burst-type fluorescent dyes. In the D-pi-A-pi-D-type dyes 1-6, a central BTD core was connected with two terminal N,N-disubstituted amino groups via various pi-conjugated spacers. The star-burst-type dyes 8 and 10 have a three-branched structure composed of a central core (benzene core in 8 and triphenylamine core in 10) and three triphenylamine-containing BTD branches. All the BTD-based dyes displayed intense orange-red color fluorescence in a region of 550-689 nm, which was obtained by single-photon excitation with good fluorescent quantum yield up to 0.98 as well as by two-photon excitation. Large two-photon absorption (TPA) cross-sections (110-800 GM) of these BTD dyes were evaluated by open aperture Z-scan technique with a femtosecond Ti/sapphire laser. The TPA cross-sections of D-pi-A-pi-D-type dyes 2-6 with a benzene, thiophene, ethene, ethyne, and styrene moiety, respectively, as an additional pi-conjugated spacer are about 1.5-2.5 times larger than that of 1c with only a benzene spacer. The TPA cross-sections significantly increased in three-branched star-burst-type BTDs 8 (780 GM) with a benzene core and 10 (800 GM) with a triphenylamine core, which are about 3-5 times larger than those of the corresponding one-dimensional sub-units 9 (170 GM) and 11 (230 GM), respectively. The ratios of sigma/e(pi) between three-branched and one-dimensional dyes were 6.5:3.8 (for 8 and 9) and 6.0:4.0 (for 10 and 11), which are larger than those predicted simply on the basis of the chromophore number density (1:1), according to a cooperative enhancement of the two-photon absorbing nature in the three-branched system.     

Benzothiadiazole-Substituted Aza-BODIPY Dyes: Two-Photon Absorption Enhancement for Improved Optical Limiting Performances in the Short-Wave IR Range

Aza-boron dipyrromethenes (aza-BODIPYs) presenting a benzothiadiazole substitution on upper positions are described. The strong electron-withdrawing effect of the benzothiadiazole moiety permits enhancement of the accepting strength and improves the delocalization of the aza-BODIPY core to attain a significant degree of electronic communication between the lower donating groups and the upper accepting groups. The nature of the intramolecular charge transfer is studied both experimentally and theoretically. Linear spectroscopy highlighted the strongly redshifted absorption and emission of the synthesized molecules with recorded fluorescence spectra over 1000 nm. Nonlinear optical properties were also investigated. Strong enhancement of the two-photon absorption of the substituted dyes compared with the unsubstituted one (up to 4520 GM at 1300 nm) results in an approximately 15–20 % improvement of the optical power limiting performances. These dyes are therefore a good starting point for further improvement of optical power limiting in the short-wave IR range.     

On the Road Toward More Efficient Biocompatible Two-Photon Excitable Fluorophores

Red-to-NIR absorption and emission wavelengths are key requirements for intravital bioimaging. One of the way to reach such excitation wavelengths is to use two-photon excitation. Unfortunately, there is still a lack of two-photon excitable fluorophores that are both efficient and biocompatible. Thus, we design a series of biocompatible quadrupolar dyes in order to study their ability to be used for live-cell imaging, and in particular for two-photon microscopy. Hence, we report the synthesis of 5 probes based on different donor cores (phenoxazine, acridane, phenazasiline and phenothiazine) and the study of their linear and non-linear photophysical properties. TD-DFT calculations were performed and were able to highlight the structure-property relationship of this series. All these studies highlight the great potential of three of these biocompatible dyes for two-photon microscopy, as they both exhibit high two-photon cross-sections (up to 3650 GM) and emit orange to red light. This potential was confirmed through live-cell two-photon microscopy experiments, leading to images with very high brightness and contrast.     

The Effect of Branching on the One- and Two-Photon Absorption,Cell Viability,and Localization of Cationic Triarylborane Chromophores with Dipolar versus Octupolar Charge Distributions for Cellular Imaging

Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two-photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two-photon excited fluorescence (TPEF) live-cell imaging.     

Impact of Ion-Pairing Effects on Linear and Nonlinear Photophysical Properties of Polymethine Dyes**

The two-photon absorption (2PA) and photophysics of heptamethine dyes featuring cyanine or dipolar electronic structures have been compared for the first time. The perfectly delocalized cyanine system is classically characterized by a two-photon transition matching the vibronic component of its lower energy absorption band. The dipolar species is generated by ion-pairing with a hard counterion in a non-dissociating solvent and displays significant modifications oft he optical properties, including a significant hypochromic shift of absorption, weaker emission and 2PA matching the lower energy transition, thus revealing symmetry breaking within the polymethine electronic structure.     

Benzothiazoles with tunable electron-withdrawing strength and reverse polarity: a route to triphenylamine-based chromophores with enhanced two-photon absorption

A series of dipolar and octupolar triphenylamine-derived dyes containing a benzothiazole positioned in the matched or mismatched fashion have been designed and synthesized via palladium-catalyzed Sonogashira cross-coupling reactions. Linear and nonlinear optical properties of the designed molecules were tuned by an additional electron-withdrawing group (EWG) and by changing the relative positions of the donor and acceptor substituents on the heterocyclic ring. This allowed us to examine the effect of positional isomerism and extend the structure-property relationships useful in the engineering of novel heteroaromatic-based systems with enhanced two-photon absorption (TPA). The TPA cross-sections (δ(TPA)) in the target compounds dramatically increased with the branching of the triphenylamine core and with the strength of the auxiliary acceptor. In addition, a change from the commonly used polarity in push-pull benzothiazoles to a reverse one has been revealed as a particularly useful strategy (regioisomeric control) for enhancing TPA cross-sections and shifting the absorption and emission maxima to longer wavelengths. The maximum TPA cross-sections of the star-shaped three-branched triphenylamines are ～500-2300 GM in the near-infrared region (740-810 nm); thereby the molecular weight normalized δ(TPA)/MW values of the best performing dyes within the series (2.0-2.4 GM·g(-1)·mol) are comparable to those of the most efficient TPA chromophores reported to date. The large TPA cross-sections combined with high emission quantum yields and large Stokes shifts make these compounds excellent candidates for various TPA applications, including two-photon fluorescence microscopy.     

嘧啶基双光子荧光染料的设计合成与生物成像

生理条件下光学性质稳定的双光子荧光染料在生物成像领域具有广阔的应用前景。我们使用2,4-二甲基-6-羟基嘧啶与4-(N,N-二甲氨基)苯甲醛进行缩合反应,设计合成了具有双光子荧光性质的化合物2-[(1E)-2-[4-(二甲氨基)苯基]乙烯基]-6-甲基-4(3H)-嘧啶(NHP)。通过质谱(MS)、核磁共振波谱(NMR)、紫外可见吸收光谱和荧光发射光谱等技术手段表征了其结构,研究了其光物理性质,以及外部环境改变对其发射光谱的影响。结果表明,化合物NHP的最佳吸收峰位于400 nm,最佳发射峰位于540 nm左右,且荧光发射不受金属离子、氨基酸和pH等环境因素的影响。生物实验结果表明,化合物NHP细胞毒性较小,且具有很好的活细胞和果蝇脑组织成像效果,是一种较为理想的双光子荧光生物成像染料。     

Synthesis and two-photon absorption of highly soluble three-branched fluorenylene-vinylene derivatives

Two new three-branched fluorenylene-vinylene derivatives were synthesized by triple Heck-type or Horner-Wadsworth-Emmons reactions. Their one-photon absorption and fluorescence as well as their two-photon absorption properties are reported. These compounds, which combine very high solubility in organic solvents, high fluorescence quantum yield and giant two-photon absorption cross-sections in the red-NIR region (up to 3660 GM, in the femtosecond regime) are promising candidates for both optical power limiting applications and two-photon laser scanning microscopy.     

Theoretical insight into linear optical and two-photon absorption properties for a series of N-arylpyrrole-based dyes

N-arylpyrrole-based dyes possessing excellent opto-electronic properties are promising candidates for two-photon fluorescence labeling materials. The systematic investigation of novel N-arylpyrrole derivatives is of great importance for both fine-tuning electronic spectra and designing two-photon absorption (2PA) materials. We thoroughly studied influences of the π-conjugated center and N-substituted pyrrole moieties on the linear optical and 2PA properties. Our results show that introducing N-arylpyrrole produces bathochromic-shifts of the absorption and emission bands and an enhancement of the 2PA cross section (δ(max)), demonstrating that the electron-rich pyrrole moieties can efficiently increase intramolecular charge transfer. Substitution of fluorenyl with benzothiadiazole produces the lower energy gap, higher emission lifetime, red-shift of 2PA spectra and larger δ(max). The absorption and emission bands are highly dependent on the nature of the active building blocks. The aromatic rings attached to pyrrole can modulate the absorption peaks in the high energy and thus subtly modify the electronic properties.     

Polyethylene glycol-functionalized benzylidene cyclopentanone dyes for two-photon excited photodynamic therapy

A series of polyethylene glycol-functionalized benzylidene cyclopentanone dyes with varying lipid/water partition coefficients were synthesized in high yields by a simple process. Detailed characterization and systematic studies of these molecules, including linear and nonlinear photophysical properties, reactive oxygen yields, and in vitro photodynamic therapy (PDT) activities, were conducted. Four of these dyes exhibited good solubility in PBS (>2 mg ml(-1), which is sufficient for clinical venous injection), high reactive oxygen yields, large two-photon absorption and low dark toxicity, under the therapy dosage. Among them, two dyes could be absorbed efficiently by human rectal cancer 1116 cells, and presented strong two-photon excited PDT activity in in vitro cell experiments.