Two-photon Action Cross Section Research Articles

Ultrafast supercontinuum sculpting for two-photon spectroscopy and microscopy of ratiometric fluorescent indicators

We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy.

Effect of Two-Photon Excitation to 8-Azacoumarin Derivatives as Photolabile Protecting Groups.

An improvement of the two-photon excitation was achieved using 8-azacoumarin-type caged compounds, which showed large values of the two-photon uncaging action cross-section (δu >0.1 Goeppert-Mayer (GM)). In particular, the 7-hydroxy-6-iodo-8-azacoumarin (8-aza-Ihc)-caged compound showed an excellent uncaging action cross-section value (δu = 1.28 GM). Therefore, 8-azacoumarin-type photolabile protecting groups (PPGs) can be used as two-photon excitation sources.

Rigidify styryl-pyridinium dyes to benzo[h]coumarin-based bright two-photon fluorescent probes for cellular bioimaging.

Fluorescence imaging of organelles at the cellular level is important for studying biological processes. The development of a highly emissive fluorescent probe that operates under a suitable excitation light source is a key step in high-quality fluorescence imaging. For long-term, high-fidelity fluorescence imaging of mitochondria-related cellular processes using two-photon microscopy and stimulated emission depletion microscopy, we developed a new benzocoumarin-based cationic fluorescent probe (BS-CN) that is far-red emitting, water-soluble, photostable, and very bright in cells. BS-CN showed a remarkably high quantum yield of 0.35 and a large two-photon excited fluorescence action cross-section of 76 GM, enabling the long-term tracking of mitochondria in live cells. In addition, BS-CN exhibited a certain affinity for RNA and stained nucleoli in fixed cells. A comparative assessment of the photophysical properties and bioimaging performance of benzo[h]coumarin-pyridinium and the structurally similar styryl-pyridinium (BS-MN) clearly indicated the importance of structural rigidity for fluorescence efficiency.

Probing the deep brain: Enhanced multi-photon imaging by aggregation-induced emission luminogens via nanocrystallization

The latest requirement of optical imaging is to visualize vital, thick, and complex biological tissues in real-time and minimum-invasive mode. Among diverse available diagnostic tools, multi-photon imaging (MPI) with long wavelength excitation and exceptional lateral resolution has been a perfect option. However, the existing palette of MPI contrast agents either suffers from sub-optimal imaging performance or non-negligible risk in biosafety, exploration of suitable probes is thus of great significance. To address these concerns, in this work, pure organic nanocrystals (NCs) were developed based on aggregation-induced emission luminogens (AIEgens) (DPTA-BT) with an ultrasound-assisted method. Compared to the amorphous DPTA-BT nanoparticles (NPs), NCs with orderly arranged molecules exhibited highly enhanced nonlinear optical signals, including up to 15.35 folds of two-photon action cross-section (2PACS) and up to 4.11 folds of three-photon action cross-section (3PACS). The combination of MPI and DPTA-BT NCs has contributed to a 1900 μm imaging depth of vasculature after craniotomy and a 765 μm imaging depth through the intact skull. The safe use of DPTA-BT NCs was verified by the in vitro and in vivo biosafety assessment. This work is thus expected to encourage more innovation in the development of organic contrast agents for MPI.

Sensitized 1-Acyl-7-nitroindolines with Enhanced Two-Photon Cross Sections for Release of Neurotransmitters.

Precise photochemical control, using two-photon excitation (2PE), of the timing and location of activation of glutamate is useful for studying the molecular and cellular physiology of the brain. Antenna-based light harvesting strategies represent a general method to increase the sensitivity to 2PE of otherwise insensitive photoremovable protecting groups (PPGs). This was applied to the most commonly used form of "caged" glutamate, MNI-Glu. Computational investigation showed that a four- or six-carbon linker attached between the 4-position of thioxanthone (THX) and the 4-position of the 5-methyl derivative of MNI-Glu (MMNI-Glu) would position the antenna and PPG close to one another to enable Dexter energy transfer. Nine THX-MMNI-Glu conjugates were prepared and their photochemical properties determined. Installation of the THX antenna resulted in a red shift of the absorption (λmax = 385-405 nm) along with increased quantum yield compared to the parent compound MNI-Glu (λmax = 347 nm). The THX-MMNI-Glu conjugate with a four-carbon linker and attachment to the 4-position of THX underwent photolysis via 1PE at 405 and 430 nm and via 2PE at 770 and 860 nm, yielding glutamate. The two-photon uncaging action cross section (δu) was 0.11 and 0.29 GM at 770 and 860, respectively, which was greater than for MNI-Glu (0.06 and 0.072 GM at 720 and 770 nm, respectively). The THX sensitizer harvested the light via 2PE and transferred its resulting triplet energy to MMNI-Glu. Release of glutamate through 2PE at 860 nm from the compound (100 μM) activated iGluSnFR, a genetically encoded, fluorescent glutamate sensor, on the surface of cells in culture, portending its usefulness in studies of neurophysiology in acute brain slice.

Optoelectronic response of semiconductor CsPb1‒xCdxBr3 nanocrystals: Role of Cd element

Cd element alloyed perovskite nanocrystals is an effective strategy to improve the photovoltaic performance. However, it still remains necessary to further explore the effect of Cd element in photo-physics and opto-eletronic performance. Here, the optoelectronic characteristics of Cd alloyed CsPbBr3 nanocrystals (CsPb1‒xCdxBr3 NCs) is investigated systemically. With the incorporation of Cd element, the crystal structure passes through from cubic (x = 0.0) to hexagonal phase (x = 0.85). The linear absorption cross sections at 400 nm monotonically decrease with x increasing, while the two-photon absorption action cross section at 800 nm increases to the maximum of 5.7 × 105 GM at x = 0.10. Meanwhile, the thermal activation model perfectly explains the optoeletronic performance of prototype photodetectors based on CsPb1‒xCdxBr3 NCs whose photo-current and photo-responsivity reach the maximum at x = 0.27. Finally, the corresponding primitive flexible photodetector shows negligible change with the device bending. Our results offer a deep insight into the strategy of Cd element alloyed into perovskite nanocrystals and broaden their application in nonlinear optical imaging or photo-detector.

Evolving a novel red-emitting two-photon dye with optically tunable amino group for monitoring the degree of hypoxia during liver fibrosis

Two-photon imaging has attracted increasing attention owing to its deep tissue imaging capabilities. Therefore, many fluorophores have been developed to satisfy its requirements. However, long-wavelength emission fluorophores with an optically tunable group are rarely developed. In this study, two long-wavelength emission fluorophores with an optically tunable amino group were successfully developed by introducing strong electron acceptor and large conjugated group to the TPQL dye. TPCO2 displayed a bright red emission (λem = 638 nm, Φ = 0.15) together with high two-photon action cross section and good water solubility, which enabled higher signal-to-background ratios and deep tissue imaging. The proof-of-concept probe (TPCONO2) was successfully applied to the high signal-to-background ratio imaging of nitroreductase in liver fibrosis, further realizing diagnosis of the degree of hypoxia during liver fibrosis.

Organelle-specific blue-emitting two-photon probes for calcium ions: Combination with green-emitting two-photon probe for simultaneous detection of proton ions

In this study, we developed organelle-specific blue-emitting two-photon (TP) probes for Ca2+ (BCa-1, BCa-2mito, and BCa-3mem), with absorption maxima (λmax) at 350–358 nm, emission maxima (λfl) at 464–466 nm, and TP action cross-section (Φδmax) values of 55–70 × 10−50 cm4s/photon, in the presence of excess Ca2+ at 750 nm. Moreover, the probes had dissociation constants of 0.18, 2.7, and 100 μM, respectively, which are appropriate values for sensing Ca2+ in the cytoplasm, mitochondria, and plasma membrane, respectively. The measurements were conducted using a calcium calibration buffer (10 mM 3-[N-morpholino]propanesulfonic acid and 100 mM KCl) at pH 7.2. The TP microscopy results revealed that the probes could facilitate the real-time detection of Ca2+ in the cytoplasm, mitochondria, and plasma membranes of live cells and tissues. Additionally, we developed a green-emitting TP probe for H+ (FHEt-1lyso) with λmax = 359 nm, λfl = 571 nm, and Φδmax = 70 × 10−50 cm4s/photon at pH 4.3 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M tris[hydroxymethyl]aminomethane, and 0.1 M KCl); this probe could detect H+ in the lysosomes. Using BCa-1 and FHEt-1lyso, it was possible to simultaneously monitor the changes in cytosolic Ca2+ and lysosomal H+ concentrations in live cells and tissues using dual-color TP microscopy in real time. When used with TP probes emitting wavelengths of green light or longer, these blue-emitting Ca2+ probes can be used to investigate the physiological role of Ca2+ in cellular organelles as well as the crosstalk between Ca2+ and other metal ions in specific organelles.

A porphyrin-triazatruxene dyad for ratiometric two-photon fluorescent sensing of intracellular viscosity.

By combining an electron-rich triazatruxene unit (TAT) to an electron-deficient zinc porphyrin fluorophore (ZnPor) via an ethynyl bridge, a new two-photon fluorescent viscosity rotor (TAT-ZnPor) with typical donor-π-acceptor (D-π-A) electronic configuration was developed for the ratiometric two-photon fluorescent detection of intracellular viscosity. The TAT-ZnPor dyad exhibited highly improved fluorescence quantum yield (Φem = 0.40) and two-photon absorption cross-section (δTPA = 811 GM) in comparison to the individual components. In the methanol/glycerol system, TAT-ZnPor showed sensitive fluorescence responses toward the change of viscosity. Upon elevating the viscosity from 0.59 to 947 cp, the blue emission band around 410 nm gradually enhanced, while the red band at 647 nm concomitantly quenched, leading to a remarkable intensity ratio (I410/I647) change from 0.70 to 81 (116-fold). TAT-ZnPor also displayed good cell imaging performance under one- and two-photon excitation, and strong mitochondria targeting ability in living cells, thus was successfully applied in detecting the change of mitochondrial viscosity during the nystatin-induced degeneration.

Stable and wide-wavelength tunable luminescence of CsPbX3 nanocrystals encapsulated in metal–organic frameworks

The obtained ZJU-28⊃CsPbX3 crystals exhibit giant two-photon action cross-section and photostability enhancement due to the effects of confinement, passivation and separation derived from ZJU-28 frameworks.

Novel two-photon absorption compounds with different organic cations: facile synthesis, photophysical properties, detection of viscosity, and selective imaging of the endoplasmic reticulum in living cells.

Three novel imidazole-based two-photon absorption compounds bearing different organic cations (1PIPy, 2PIQu, and 3PIIm) were facilely synthesized and fully characterized by 1H NMR, 13C NMR, FT-IR, and HRMS. The linear and nonlinear photophysical properties of the target compounds were systematically investigated in various solvents, supplemented with the density functional theory calculations to shed light on their structure-property relationships. The maximum two-photon action cross-sections (Φ × δmax) were determined to be 22.4-98.2 (CH2Cl2), 9.6-41.3 (DMF), and 3.9-11.8 (H2O) GM. It is found that 3PIIm shows significant viscosity sensitivity with a sharp 27-fold increase in fluorescence intensity. Its fluorescence intensity also exhibits a linear relationship with the viscosity of the media in a logarithmic plot. The Φ × δmax value of 3PIIm in highly viscous glycerol was found to be 107.5 GM. Cytotoxicity tests indicate that these compounds have relatively low cytotoxicity. All the target compounds were successfully characterized by one- and two-photon fluorescence imaging in living cells. The colocalization experiments reveal that 1PIPy and 3PIIm are specially located in the endoplasmic reticulum (ER) with the Pearson's coefficients above 0.90. 3PIIm can also monitor the fluctuation of ER viscosity during etoposide-induced apoptosis.

A novel red-emitting two-photon fluorescent probe for imaging nitroreductases in cancer cells and tumor tissues with hypoxia conditions

Nitroreductases (NTRs) are overexpressed in many cancer cells especially in hypoxia conditions. They have been acted as an essential diagnosis and treatment targets for cancers. In order to detecting NTRs with high signal to background ratios using two-photon manner, it is necessary to explore effective two-photon fluorescent probes with long emission wavelengths. Here, we designed a novel red-emitting two-photon fluorescent probe P1 for tracing NTRs. P1 was constructed based on a two-photon fluorogen P1-OH with red-emission and a NTRs recognition site p-nitrobenzene. In the presence of NTRs, P1 showed strong fluorescence emission at 647 nm. P1 had high selectivity and sensitivity with NTRs (LOD is 0.00245 U/L). Importantly, two-photon action cross section experiments showed that P1 had favorable two-photon property encountering with NTRs and NADH. Furthermore, under hypoxia conditions, P1 has succeeded in imaging NTRs in cancer cells under one- and two-photon manners with hypoxia manner. Besides, by means of three-dimensional high-resolution manner, tumor tissues imaging has been achieved using P1 with two-photon manner. Probe P1 with red-emitting two-photon property may a potential molecular tool for detecting NTRs in living biological systems.

Cooperation of ESIPT and ICT Processes in the Designed 2-(2'-Hydroxyphenyl)benzothiazole Derivative: A Near-Infrared Two-Photon Fluorescent Probe with a Large Stokes Shift for the Detection of Cysteine and Its Application in Biological Environments.

A rationally designed near-infrared two-photon fluorescent probe (SDP-A) for selectively detecting cysteine (Cys) has been developed based on a newly designed conjugation-enhanced 2-(2'-hydroxyphenyl)benzothiazole derivative as the fluorophore, an acrylate moiety as the Cys reaction site, and an N-methylpyridinium scaffold as both the unit of organelle targeting and improving water solubility. The probe SDP-A alone essentially emitted no fluorescence, whereas it achieved a superb near-infrared fluorescence emission (713 nm) enhancement within 15 min with a significant Stokes shift (302 nm) in the presence of Cys. The photoluminescence mechanism of the probe SDP-A toward Cys was modulated by excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) processes. It exhibited high selectivity and sensitivity (LOD = 102 nM) for monitoring Cys over other analytes such as Hcy/GSH/H2S owing to a specific conjugate addition-cyclization reaction between Cys and the acrylate moiety. More importantly, the released fluorophore SDP exhibits elevated quantum yields (1.52-18.17%) in different solvents and strong two-photon excited fluorescence with a sizeable two-photon action cross-section (Φ) of 213.5 GM at 820 nm in acetonitrile-PBS medium, which is highly desirable for two-photon fluorescence imaging of the living samples. Therefore, SDP-A was successfully applied to the imaging of Cys in live cells, zebrafish, mouse brain, and abdominal cavity down to a depth of more than 200 μm using a one/two-photon fluorescence microscope.

Novel yellow- to red-emitting fluorophores: Facile synthesis, aggregation-induced emission, two-photon absorption properties, and application in living cell imaging

Four novel yellow-to red-emitting fluorophores with a 1-(2-hydroxyethyl) pyridinium core and different electron-donating terminal moieties (N-methylcarbazole, N,N-dibutylbenzenamine, N-methylpyrrole, and 2-methylfuran) were designed and facilely synthesized via the one-step Knoevenagel condensation. All these compounds (HPs) were characterized by 1H NMR, 13C NMR, and HRMS. Their photophysical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. And the density functional theory calculations were conducted to analyze the electronic structures of HPs. The two-photon absorption cross-sections (δ) values of HPs measured by the Z-scan technique were determined to be as large as 1354 (1HP-CZ), 4462 (2HP-BA), 836 (3HP-PR), and 2944 GM (4HP-FU) in DMSO. The two-photon action cross-sections (Φ × δ) values of 1HP-CZ and 2HP-BA in H2O measured by the two-photon induced fluorescence method were about 50 GM. 1HP-CZ, 3HP-PR, and 4HP-FU also exhibited good water solubility. Meanwhile, it was found that 2HP-BA exhibited notable aggregation-induced emission characteristic in DMSO/H2O mixture. The aggregate particle size distribution of 2HP-BA was measured by the dynamic light scattering method, and the aggregation characteristic of 2HP-BA was observed by the transmission electron microscopy. Besides, 2HP-BA also exhibited red emission. Then, HPs with relatively low cytotoxicity were used for one- and two-photon excited fluorescence imaging in living HepG2 cells. The results indicate that HPs are potential candidates in the bioimaging field due to their photophysical properties and biocompatibility.

Turning on two-photon activity over N∧N∧N cyclometalated Pt(II) complex by introducing flexible chains

The rational design and fabrication of two-photon excited fluorescent probes for noninvasive bioimaging with deeper tissue penetration and higher spacial resolution is a high-priority target yet grand challenge. To this end, herein, a novel amphiphilic N∧N∧N cyclometalated Pt(II) complex (Pt–N1) was designed and synthesized. Benefiting from its flexible chains and abundant electronic structure, it displayed excellent aggregation-induced emission (AIE) behavior under physiological conditions due to its self-assembly process with the proportion of water increased. As a result, Pt–N1 exhibited considerable two-photon action cross-section in aggregation state, suggesting its potential application in bioimaging.

Controlling Two-Photon Action Cross Section by Changing a Single Heteroatom Position in Fluorescent Dyes.

The optimization of nonlinear optical properties for “real-life” applications remains a key challenge for both experimental and theoretical approaches. In particular, for two-photon processes, maximizing the two-photon action cross section (TPACS), the figure of merit for two-photon bioimaging spectroscopy, requires simultaneously controlling all its components. In the present Letter, a series of difluoroborates presenting various heterocyclic rings as an electron acceptor have been synthesized and their absorption, fluorescence, photoisomerization, and two-photon absorption features have been analyzed using both experimental and theoretical approaches. Our results demonstrate that the TPACS values can be fine-tuned by changing the position of a single heteroatom, which alters the fluorescence quantum yields without changing the intrinsic two-photon absorption cross section. This approach offers a new strategy for optimizing TPACS.

Covalent Organic Framework for Efficient Two-Photon Absorption

Summary We report the collective alignment of molecular chromophores in the backbone of covalent organic frameworks (COFs) as a new chemical approach to construct efficient two-photon absorption (2PA) materials. Six imine COFs were synthesized from aldehyde-terminated triarylamine linkers and p-phenylenediamine. Attainment of these COFs in single-crystal form revealed the underlying layered structures and precise spatial arrangements of the constituent chromophores including an unusual serrated packing between the layers. Coherent orientation of chromophores within each layer made possible collective alignment of their transition dipoles, while the serrated packing between the layers maximized the crystal anisotropy. As a result, the full potential of molecular chromophores for 2PA was developed. Up to 110-fold enhancement of two-photon action cross-section (2PACS) was achieved for the COFs relative to their constituents, with one member exhibiting a high 2PACS value of 8,756 GM/chromophore-standing among the best molecule-based 2PA materials.

One- and two-photon electron-transfer induced uncaging of coumarin from cinnamate-capped CdSe quantum dots

It has been previously shown that visible-light excitation of CdSe quantum dots (QD) functionalized with a cinnamate ligand (4-Diethylamino-2-hydroxythiocinnamate; 4DEATC) can induce uncaging of a fluorescent coumarin molecule (7-Diethylaminocoumarin; 7DEAC). In the current study, the quantum yield, QY, for this photochemical uncaging process is determined. In addition, the results of visible-light excitation of cinnamate-capped CdSe and cinnamate-capped CdSe/ZnS QDs are compared. The uncaging of coumarin is observed only for the functionalized core CdSe QDs, and not for CdSe/ZnS core-shell QDs. These results strongly support that the uncaging of coumarin from cinnamate-capped QDs occurs via an electron-transfer step from the QD to the cinnamate ligand, and the large band gap of the ZnS shell blocks electron transfer from CdSe core to the ligand. In addition, it is demonstrated that coumarin uncaging from cinnamate-capped CdSe can be induced using pulsed NIR (1064 nm) excitation of the QD via a two-photon process. The two-photon absorbance cross section and two-photon absorbance action cross section are estimated by combining the results of the one- and two-photon uncaging experiments.

Methoxy-Substituted Nitrodibenzofuran-Based Protecting Group with an Improved Two-Photon Action Cross-Section for Thiol Protection in Solid Phase Peptide Synthesis.

Photoremovable caging groups are useful for biological applications because the deprotection process can be initiated by illumination with light without the necessity of adding additional reagents such as acids or bases that can perturb biological activity. In solid phase peptide synthesis (SPPS), the most common photoremovable group used for thiol protection is the o-nitrobenzyl group and related analogues. In earlier work, we explored the use of the nitrodibenzofuran (NDBF) group for thiol protection and found it to exhibit a faster rate toward UV photolysis relative to simple nitroveratryl-based protecting groups and a useful two-photon cross-section. Here, we describe the synthesis of a new NDBF-based protecting group bearing a methoxy substituent and use it to prepare a protected form of cysteine suitable for SPPS. This reagent was then used to assemble two biologically relevant peptides and characterize their photolysis kinetics in both UV- and two-photon-mediated reactions; a two-photon action cross-section of 0.71-1.4 GM for the new protecting group was particularly notable. Finally, uncaging of these protected peptides by either UV or two-photon activation was used to initiate their subsequent enzymatic processing by the enzyme farnesyltransferase. These experiments highlight the utility of this new protecting group for SPPS and biological experiments.

Two-Photon Excitable Photoremovable Protecting Groups Based on the Quinoline Scaffold for Use in Biology.

Photoremovable protecting groups (PPGs) are powerful tools for physiological studies, harnessing light as an on/off switch to provide tight spatio-temporal control over the release of biological effectors through two-photon excitation (2PE) in tissue culture and whole-animal studies. We carried out a series of systematic structural modifications to the (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ) chromophore to conduct an SAR study with the aim of enhancing its photochemical properties, especially its two-photon uncaging action cross section (δu). The best results were obtained when substituents were added at the C4 position, which improved δu for release of acetate up to 7-fold, while retaining all the other excellent properties of the CyHQ PPG, including high quantum yield (Φu), low susceptibility to spontaneous hydrolysis in the dark, and good aqueous solubility. Hammett correlation analysis suggested that photolysis efficiency is favored by electron-rich substituents at C4, giving important insights into the mechanism of the photolysis reaction. The four best CyHQ derivatives were used to mediate the efficient release of homopiperonylic acid in high yield under simulated physiological conditions. Our efforts have led to the development of 2PE-sensitive PPGs with remarkable δu values (up to 2.64 GM), excellent quantum yields (up to 0.88), and high-yielding effector release (up to 92%).