Photophysics of a series of efficient fluorescent pH probes for dual-emission-wavelength measurements in aqueous solutions

This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission.     

6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection

New ratiometric two-photon fluorescent probes are developed from 6-substituted quinolines for biological Zn(2+) detection. They show large red shifts and good ratiometric responses upon Zn(2+) binding. They also exhibit high ion selectivities and large two-photon absorption cross sections at nearly 720 nm. Because the new probes are cell-permeable, they can be used to detect intracellular zinc flux under two-photon excitation.     

双光子荧光探针研究及其应用*

双光子荧光显微成像兼具诸如近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等特点而显著地优于单光子荧光显微成像,为生命科学研究提供了更为锐利的工具。而用于像离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面研究的双光子荧光探针,是实现成像的关键。双光子荧光探针的研究旨在促进双光子荧光显微镜应用的发展,促进生命科学、医学科学的快速发展,同时也带动双光子荧光探针所隶属的化学这一学科的发展。因此对双光子荧光探针的研究具有重要的理论和实践意义。该文综述了双光子荧光显微成像的优点、双光子荧光探针设计的原理及双光子荧光探针在离子分析方面的应用,并展望了这类荧光探针的发展趋势与应用前景。     

The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue

6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.     

NIR-Ⅱ Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scattering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emission(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggregated dots.Herein,we report two-photon fluorescence lifetime microscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×10⁴GM at 1040 nm.Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser.The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17μm were still observed with a nice signal-background ratio at the depth of 750μm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.     

Strong modulation of two-photon excited fluorescence of quadripolar dyes by (de)protonation

Two quadripolar dyes have been designed and synthesized that present large cross sections for two-photon excitation and whose fluorescence responds strongly to (de)protonation. These dyes are considered as prototypes of molecular pH probes for multiphoton fluorescence microscopy.     

o-nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses and uncaging cross-sections for one- and two-photon excitation

We evaluated the o-nitrobenzyl platform for designing photolabile protecting groups with red-shifted absorption that could be photolyzed upon one- and two-photon excitation. Several synthetic pathways to build different conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position, are reported. Relative to the reference 4,5-dimethoxy-2-nitrobenzyl group, several o-nitrobenzyl derivatives exhibit a large and red-shifted one-photon absorption within the near-UV range. Uncaging after one-photon excitation was studied by measuring UV-visible absorption and steady-state fluorescence emission on model caged ethers and esters. In the whole series investigated, the caged substrates were released cleanly upon photolysis. Quantum yields of uncaging after one-photon absorption lie within the 0.1-1 % range. We observed that these drop as the maximum wavelength absorption of the o-nitrobenzyl protecting group is increased. A new method based on fluorescence correlation spectroscopy (FCS) after two-photon excitation was used to measure the action uncaging cross section for two-photon excitation. The series of o-nitrobenzyl caged fluorescent coumarins investigated exhibit values within the 0.1-0.01 Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields of uncaging associated with cross-sections of 1-50 GM for two-photon absorption. Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl photolabile protecting groups can be readily improved, we emphasize the difficulty in enlarging the corresponding action uncaging cross-sections in view of the observed trend of their quantum yield of uncaging.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

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有双光子吸收的金属配合物对生物分子(如pH、O₂、HClO、NO、SO₂、GSH、DNA等)的响应检测用于疾病的诊断,以及作为细胞器(如线粒体、溶酶体、脂滴、细胞核等)染料探针用于细胞内动态行为和演化过程的实时示踪研究。最后,针对金属配合物在生物分子探针以及细胞器染料等方面的应用前景进行了分析和探讨。     

Diaroyl(methanato)boron difluoride compounds as medium-sensitive two-photon fluorescent probes

This paper evaluates the use of diaroyl(methanato)boron difluoride compounds for designing efficient fluorescent probes through two-photon absorption. Three different pathways allowing for the syntheses of symmetrical and dissymmetrical molecules are reported. The stable diaroyl(methanato)boron difluoride derivatives can be easily obtained in good yields. They exhibit a large one-photon absorption that is easily tuned in the near-UV range. Their strong fluorescence emission covers the whole visible domain. In addition to these attractive linear properties, several diaroyl(methanato)boron difluoride derivatives possess significant cross sections for two-photon absorption. The derived structure-property relationships are promising for designing new generations of molecules relying on the diaroyl(methanato)boron difluoride backbone.     

Metal ion sensing novel calix[4]crown fluoroionophore with a two-photon absorption property

1,3-Alternate calix[4]arene-based fluorescent chemosensors bearing two-photon absorbing chromophores have been synthesized, and their sensing behaviors toward metal ions were investigated via absorption band shifts as well as one- and two-photon fluorescence changes. Free ligands absorb the light at 461 nm and weakly emit their fluorescence at 600 nm when excited by UV-vis radiation at 461 nm, but no two-photon excited fluorescence is emitted by excitation at 780 nm. Addition of an Al(3+) or Pb(2+) ion to a solution of the ligand causes a blue-shifted absorption and enhanced fluorescence due to a declined resonance energy transfer (RET) upon excitation by one- and two-photon processes. Addition of a Pb(2+) ion to a solution of 1.K(+) results in a higher fluorescence intensity than the original 1.Pb(2+) complex regardless of one- or two-photon excitation, due to the allosteric effect induced by the complexation of K(+) with a crown loop.     

Tracking of mercury ions in living cells with a fluorescent chemodosimeter under single- or two-photon excitation

Tracking of Hg²⁺ in solutions as well as in living cells was conducted with a fluorescent chemodosimeter by measuring the spectral shift of its fluorescence under single- or two-photon excitation. The spectral hypsochromic shifts of this chemodosimeter when reacting with Hg²⁺ were found to be about 50 nm in acetonitrile/water solutions and 32 nm in Euglena gracilis 277 living cells. This chemodosimeter shows high sensitivity and selectivity, and is not influenced by the pH values. It can signal Hg²⁺ in solutions down to the ppb range under either single-photon excitation (SPE) at 405 nm or two-photon excitation (TPE) at 800 nm. However, with low cellular chemodosimeter concentrations, the SPE spectra were disturbed by the auto-fluorescence from the native fluorophore in the cell, while the TPE spectra were still of high quality since the two-photon absorption cross section of this chemodosimeter is much larger than that of the native fluorophores in the cell.     

Two-photon uncaging with fluorescence reporting: evaluation of the o-hydroxycinnamic platform

This paper evaluates the o-hydroxycinnamic platform for designing efficient caging groups with fluorescence reporting upon one- and two-photon excitation. The model cinnamates are easily prepared in one step by coupling commercial or readily available synthons. They exhibit a large one-photon absorption that can be tuned in the near-UV range. Uncaging after one-photon excitation was investigated by 1H NMR, UV-vis absorption, and steady-state fluorescence emission. In the whole investigated series, the caged substrate is quantitatively released upon photolysis. At the same time, uncaging releases a strongly fluorescent coproduct that can be used as a reporter for quantitative substrate delivery. The quantum yield of double bond photoisomerization leading to uncaging after one-photon absorption mostly lies in the 10% range. Taking advantage of the favorable photophysical properties of the uncaging coproduct, we use a series of techniques based on fluorescence emission to measure the action uncaging cross sections with two-photon excitation of the present cinnamates. Exhibiting values in the 1-10 GM range at 750 nm, they satisfactorily compare with the most efficient caging groups reported to date. Noticeably, the uncaging behavior with two-photon excitation is retained in vivo as suggested by the results observed in living zebrafish embryos. Reliable structure property relationships were extracted from analysis of the present collected data. In particular, the careful kinetic analysis allows us to discuss the relevance of the o-hydroxycinnamic platform for diverse caging applications with one- and two-photon excitation.     

基于咔唑的双光子荧光次氯酸根探针光学性质及响应机理

采用含时密度泛函理论与响应函数理论相结合的方法,研究了两种实验室新合成的水溶性双光子荧光次氯酸根（ClO^-）探针分子HCH和HCM的单光子吸收、双光子吸收和荧光发射性质。计算结果表明,HCH和HCM分子与ClO^-反应后,生成物的光吸收和光发射性质均发生明显变化,相应的吸收和发射峰位都发生了明显蓝移,荧光强度展现出明显的增强。另外,两探针分子都具有较大的双光子吸收截面,且与ClO^-反应后,生成物的双光子吸收截面值显著增加,因此两分子均可作为性能优良的双光子荧光探针分子。此外,通过分析HCH和HCM分子与ClO^-反应前后的Mulliken电荷布居情况,从理论上证实了该系列荧光探针分子的识别机理是C＝N异构化。     

Fluorescence behavior of 7-hydroxycoumarine excited by one-photon and two-photon absorption by means of a tunable dye laser

The fluorescence spectrum of 7-hydroxycoumarine in ethanol excited by a pulsed tunable dye laser reveals different features when excitation proceeds via one-photon and two-photon absorption. In the former case the spectrum shows two peaks delayed in time by approximately 2 ns and characterized by different lifetimes. The relative intensity of these peaks is unaffected by significant changes in the dye concentration and therefore the formation of an “exciplex” species seems to be here confirmed. In the spectrum obtained by two-photon excitation the second peak at longer wavelength is absent. Results are presented for both room and liquid nitrogen temperatures as well as for other solvents such as glycerin and EPA. These results are considered to be important for the evaluation of absolute two-photon cross sections where the quantum efficiencies of one- and two-photon processes are assumed to be the same.     

双氰基二苯代乙烯型双光子荧光汞离子探针

分别以双氰基二苯代乙烯(DCS)和双[2-(2-羟乙基硫基)乙基]氨(HSA)为双光子荧光团和汞离子受体,合成了双光子荧光汞离子探针(DHg),并对其结构进行了分析.实验结果表明,DHg在甲苯、乙腈和水中的荧光量子产率(Φ)分别为0.78,0.42和0.20,对汞离子的络合常数通过单、双光子荧光滴定分别拟合为lg K=5.47±0.02和lg K=5.34±0.02.DHg在水溶液中对汞离子具有优良的选择性和高的灵敏性,可用于中性环境中汞离子的检测.DHg的双光子吸收截面(δTPA)在水溶液中高达840 GM,可用于细胞中汞离子的检测与成像.     

Facile synthesis of ultrabright luminogens with specific lipid droplets targeting feature for in vivo two-photon fluorescence retina imaging

The application of fluorescent probes for in vivo retinal imaging is of great importance, which could provide direct and crucial imaging evidence for a better understanding of common eye diseases. Herein, a group of bright organic luminogens with typical electron-donating (D) and electron-accepting (A) structures (abbreviated as LDs-BDM, LDs-BTM, and LDs-BHM) was synthesized through a simple single-step reaction. They were found to be efficient solid-state emitters with high fluorescence quantum yields of above 70% (e.g., 83.7% for LDs-BTM). Their light-emission properties could be tuned by the modulation of π-conjugation effect with methoxy groups at different substituent positions. Their resulting fluorescent nanoparticles (NPs) were demonstrated as specific lipid droplets (LDs) targeting probes with high brightness, good biocompatibility, and satisfactory photostability. LDs-BTM NPs with a large two-photon absorption cross section (σ₂ = 249 GM) were further utilized as ultrabright two-photon fluorescence (2PF) nanoprobes for in vivo retina imaging of live zebrafish by NIR excitation at an ultralow concentration (0.5 µmol/L). Integrated histological structures at the tissue level and corresponding fine details at the cellular level of the embryonic retina of live zebrafish were clearly demonstrated. This is the first report of using ultrabright LDs-targeting nanoprobes to accurately measure fine details in the retina with 2PF microscopic technique. These good results are anticipated to open up a new avenue in the development of efficient 2PF emitters for non-invasive bioimaging of living animals.     

Two-photon fluorescent probes for long-term imaging of calcium waves in live tissue

2-Acetyl-6-(dimethylamino)naphthalene-derived two-photon fluorescent Ca2+ probes (ACa1-ACa3) are reported. They can be excited by a 780 nm laser beam, show 23-50-fold enhancement in one- and two-photon excited fluorescence in response to Ca2+, emit fourfold stronger two-photon excited fluorescence than Oregon Green 488 BAPTA-1 upon complexation with Ca2+, and can selectively detect intracellular free Ca2+ ions in live cells and living tissues with minimum interference from other metal ions and membrane-bound probes. Moreover, these probes are capable of monitoring calcium waves at a depth of 120-170 microm in live tissues for 1100-4000 s using two-photon microscopy with no artifacts of photobleaching.     

Metal-ion sensing fluorophores with large two-photon absorption cross sections: aza-crown ether substituted donor-acceptor-donor distyrylbenzenes

Chromophores based on a donor-acceptor-donor structure possessing a large two-photon absorption cross section and one or two mono-aza-15-crown-5 ether moieties, which can bind metal cations, have been synthesized. The influence of Mg(2+) binding on their one- and two-photon spectroscopic properties has been investigated. Upon binding, the two-photon action cross sections at 810 nm decrease by a factor of up to 50 at high Mg(2+) concentrations and this results in a large contrast in the two-photon excited fluorescence signal between the bound and unbound forms, for excitation in the range of 730 to 860 nm. Experimental and computational results indicate that there is a significant reduction of the electron donating strength of the aza-crown nitrogen atom(s) upon metal ion binding and that this leads to a blue shift in the position as well as a reduction in the strength of the lowest-energy two-photon absorption band. The molecules reported here can serve as models for the design of improved two-photon excitable metal-ion sensing fluorophores.     

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.