Two-photon Probe Research Articles

Mitochondrial Dynamics Tracking with Two-Photon Phosphorescent Terpyridyl Iridium(III) Complexes.

Mitochondrial dynamics, including fission and fusion, control the morphology and function of mitochondria, and disruption of mitochondrial dynamics leads to Parkinson’s disease, Alzheimer’s disease, metabolic diseases, and cancers. Currently, many types of commercial mitochondria probes are available, but high excitation energy and low photo-stability render them unsuitable for tracking mitochondrial dynamics in living cells. Therefore, mitochondrial targeting agents that exhibit superior anti-photo-bleaching ability, deep tissue penetration and intrinsically high three-dimensional resolutions are urgently needed. Two-photon-excited compounds that use low-energy near-infrared excitation lasers have emerged as non-invasive tools for cell imaging. In this work, terpyridyl cyclometalated Ir(III) complexes (Ir1-Ir3) are demonstrated as one- and two-photon phosphorescent probes for real-time imaging and tracking of mitochondrial morphology changes in living cells.

A small-molecule and resumable two-photon fluorescent probe for Zn2+ based on a coumarin Schiff-base

In this study, a simple one-photon and two-photon probe, 7-(4′-(diethylamino)-2′-hydroxybenzylideneimino)-4-methyl coumarin, for Zn2+ is designed and synthesized. The sensor shows high selectivity towards Zn2+ ions with significant fluorescence intensity enhanced at 500nm, which might be attributed to the formation of 2:1 stoichiometric L–Zn complex resulting in the restricted CN isomerization and the inhibited photo-induced electron transfer. More importantly, the reversibility of the recognition process of HL is performed by adding a Zn2+ bonding agent Na2EDTA.

Two-photon fluorescent probe for peroxynitrite

Peroxynitrite (ONOO−), a strong oxidant found in biological systems, and an increase in its levels is related to numerous diseases, immune responses, and redox regulation of signaling pathways. Herein, we report a new two-photon probe that sensitively and selectively detects ONOO− among other ROS/RNSs (reactive oxygen/nitrogen species) using peroxynitrite-triggered dearylation reaction.

Real-time tracking mitochondrial dynamic remodeling with two-photon phosphorescent iridium (III) complexes

Mitochondrial fission and fusion control the shape, size, number, and function of mitochondria in the cells of organisms from yeast to mammals. The disruption of mitochondrial fission and fusion is involved in severe human diseases such as Parkinson's disease, Alzheimer's disease, metabolic diseases, and cancers. Agents that can real-time track the mitochondrial dynamics are of great importance. However, the short excitation wavelengths and rapidly photo-bleaching properties of commercial mitochondrial dyes render them unsuitable for tracking mitochondrial dynamics. Thus, mitochondrial targeting agents that exhibit superior photo-stability under continual light irradiation, deep tissue penetration and at intrinsically high three-dimensional resolutions are urgently needed. Two-photon-excited compounds employ low-energy near-infrared light and have emerged as a non-invasive tool for real-time cell imaging. Here, cyclometalated Ir(III) complexes (Ir1–Ir5) are demonstrated as one- and two-photon phosphorescent probes for the real-time imaging and tracking of mitochondrial fission and fusion. The results indicate that Ir2 is well suited for two-photon phosphorescent tracking of mitochondrial fission and fusion in living cells and in Caenorhabditis elegans (C. elegans). This study provides a practical use for mitochondrial targeting two-photon phosphorescent Ir(III) complexes.

An organic dye with very large Stokes-shift and broad tunability of fluorescence: Potential two-photon probe for bioimaging and ultra-sensitive solid-state gas sensor

Light-emitting nonlinear optical molecules, especially those with large Stokes shifts and broad tunability of their emission wavelength, have attracted considerable attention for various applications including biomedical imaging and fluorescent sensors. However, most fluorescent chromophores have only limited potential for such applications due to small Stokes shifts, narrow tunability of fluorescence emissions, and small optical nonlinearity in highly polar solvents. In this work, we demonstrate that a two-photon absorbing stilbene chromophore exhibits a large two-photon absorption action cross-section (ηδ = 320 GM) in dimethylsulfoxide (DMSO) and shows broad fluorescence tunability (125 nm) by manipulating the polarity of the surrounding medium. Importantly, a very large Stokes shift of up to 227 nm is achieved in DMSO. Thanks to these features, this chromophore can be utilized as a two-photon probe for bioimaging applications and in an ultrasensitive solid-state gas detector.

A two-photon ratiometric ESIPT probe for the discrimination of different palladium species and its application in bioimaging.

A highly selective ratiometric ESIPT fluorescent probe has been designed for the detection of palladium species of all the typical oxidation states (0, +2, +4). The probe exhibits a ratiometric fluorescence response towards the palladium species in about 20 min at room temperature based on the excited state intramolecular proton transfer (ESIPT) process. Moreover, the probe can discriminate different palladium species (Pd0, Pd2+, Pd4+) under different detection conditions. Furthermore, the probe can be used to quantitatively detect palladium in drinking water with a low detection limit (15.6 nM, 1.66 ppb). Additionally, it could be used as a two-photon palladium probe for bioimaging with low cytotoxicity and autofluorescence.

4-tert-butylphenoxy substituted phthalocyanine with RGD motif as highly selective one-photon and two-photon imaging probe for mitochondria and cancer cell

An unsymmetrical phthalocyanine based one- and two-photon fluorescence imaging probe that substituted with 4-tert-butylphenoxy and RDGyK moieties was developed and characterized by UV-vis and high-resolution MALDI-TOF/MS. The conjugate is non-aggregated in [Formula: see text],[Formula: see text]-dimethylformamide, with relatively weak fluorescence emission ([Formula: see text] 0.023) and high singlet oxygen quantum yield ([Formula: see text] 0.55). Conjugation of the cyclic peptide sequence c(RGDyK) can enhance the cellular uptake towards the DU145 and PC3 cells. While the fluorescence is greatly enhanced in mitochondria, the conjugate is non-cytotoxicity either in dark or upon exposure to red-light with dose up to 12 J.cm[Formula: see text]. The results suggest that this conjugate is a promising multifunctional imaging probe for mitochondria and cancer.

A ratiometric two-photon probe for quantitative imaging of mitochondrial pH values.

Mitochondrial pH (pHmito) is known to be alkaline (near 8.0) and has emerged as a potential factor for mitochondrial function and disorder. We have developed a ratiometric two-photon probe (CMP1) for quantitative analysis of pHmito in live cells and tissues. This probe is designed to function by controlling the intramolecular charge transfer from 2-naphthol, having an ideal pKa value (7.86 ± 0.05) in the cells to monitor pHmito. This transition results in a marked yellow to red emission color change in response to pH alterations from 6.0 to 9.0. CMP1 exhibits easy loading, selective and robust staining ability of mitochondria, low cytotoxicity, and bright two-photon excited fluorescence in situ, thereby allowing quantitative imaging of the pHmito in live cells and tissues. The ratiometric TPM imaging clearly reveals that subcellular distribution of the pHmito values is heterogeneous, with the pHmito values in the perinuclear region being higher than those at the periphery of the cells. The changes of pHmito values on carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and autophagic processes were also investigated along with their morphological alterations at specific subcellular positions. We also used CMP1 to visualize the pHmito values of Parkinson's disease model astrocytes as well as living hippocampal tissues. Our results demonstrate that CMP1 will be useful as a quantitative imaging probe to study pHmito in biomedical research.

Photo-click construction of a targetable and activatable two-photon probe imaging protease in apoptosis.

A photo-click reaction was used as an efficient method to construct two-photon fluorescent probes bearing two functional peptides for targeting and for protease cleavage respectively. The activatable two-photon probe constructed by this method was applied to two-photon imaging of caspase-3 both in cellular apoptosis and in tumor tissue.

A versatile two-photon fluorescent probe for ratiometric imaging E. coliβ-galactosidase in live cells and in vivo.

We have described the design, synthesis, spectroscopy and biological applications of NI-βGal, a versatile fluorescent probe to detect E. coliβ-galactosidase in live cells and mice sensitively and directly, which holds great promise for its application in biomedical research such as gene therapy for cancer in the future.

A carboxylesterase-selective ratiometric fluorescent two-photon probe and its application to hepatocytes and liver tissues.

Carboxylesterases (CEs) are widely distributed enzymes in the human body that catalyze hydrolysis of various endogenous and exogenous substrates. They are directly linked to hepatic drug metabolisms and steatosis, and their regulations are important issues in pharmacological and clinical applications. In this work, we have developed an emission ratiometric two-photon probe (SE1) for quantitatively detecting CE in situ. This probe is based on a translation of intramolecular charge transfer character upon reaction with CE. It shows a sensitive blue-to-yellow emission change in response to human CE activity, easy loading into cells, insensitivity to pH and other metabolites including ROS and RNS, high photostability, and low cytotoxicity. Using live hepatocytes and liver tissues, we found that ratiometric two-photon microscopic imaging with SE1 is an effective tool for monitoring CE activities at the subcellular level in live tissues. This probe will find useful applications in biomedical research, including studies of hepatic steatosis and drug developments.

Real-time monitoring of vesicle pH in an endocytic pathway using an EGF-conjugated two-photon probe.

Herein, we developed a ratiometric two-photon probe (BHS3-EGF), derived from a pH sensitive dye and epidermal growth factor (EGF), for real-time monitoring and quantitative analysis of acidic luminal pH values during endocytic pathway activity. Two-photon microscopy imaging with BHS3-EGF allows the quantitative analysis of pH distributions of single vesicles and their dynamics in receptor-mediated endocytosis in real-time.

Graphene quantum dots conjugated with polymers for two-photon properties under two-photon excitation.

Few studies have investigated the two-photon properties of graphene quantum dots (GQDs) and GQD-conjugated polymers. The results of the present study revealed that conjugated polymers containing nitrogen and sulfur atoms caused higher quantum confinement of emissive energy to be trapped on the surface of nanomaterials, resulting in a high-photoluminescence quantum yield and notable two-photon properties. Additionally, the nanomaterials generated no reactive oxygen species-dependent oxidative stress on cells and served as promising two-photon contrast probes.

Elucidating the cellular uptake mechanism of aptamer-functionalized graphene-isolated-Au-nanocrystals with dual-modal imaging.

Elucidating the endocytosis and metabolism of nanoparticles in cells could improve the diagnostic sensitivity and therapeutic efficiency. In this work, we explore the cellular uptake mechanism of a biocompatible nanocrystal nanostructure, graphene-isolated-Au-nanocrystals (GIANs), by monitoring the intrinsic Raman and two-photon luminescence signals of GIANs in live cells. Aptamers functionalized on the GIAN nanostructure through simple, but strong, π-π interactions entered the cells through a clathrin-dependent pathway, while unmodified GIANs mainly entered the cells through a caveolae-mediated endocytosis pathway. Thus, it can be concluded that the mechanism of cellular uptake in these graphene-isolated-Au-nanocrystal nanostructures is determined by the presence or absence of aptamer modification.

A new water-soluble heteronuclear PdII–AuI pincer complex as two-photon luminescent probe for biological Co2+ detection

A new water-soluble heteronuclear PdII–AuI pincer complex was synthesized and investigated for biological Co2+ detection.

Styrylpyridine salts-based red emissive two-photon turn-on probe for imaging the plasma membrane in living cells and tissues.

Based on styrylpyridine salts, a small-molecule red emitting membrane probe with large two-photon absorption cross-section has been synthesized. As a molecular rotor, it enables exclusive lighting up of the plasma membrane in live cells and particular tissues. This probe has the potential to be a powerful tool for bioimaging.

A water-soluble colorimetric two-photon probe for discrimination of different palladium species and its application in bioimaging.

A novel water-soluble colorimetric and fluorescent palladium probe with excellent selectivity and sensitivity has been designed. Notably, based on a palladium triggered terminal allyl ether cleavage reaction, the probe could detect and discriminate Pd(0) and Pd(2+)/Pd(4+) in about 2.5 min at room temperature with a low detection limit (0.29 ppb) and significant colour change (from light yellow to pink). The probe could serve as an excellent "naked-eye" colorimetric probe for selective and quantitative determination of palladium in aqueous solutions. Moreover, it could be used as a two-photon palladium probe for in vitro/vivo and three-dimensional imaging with low cytotoxicity and autofluorescence.

NIR in, far-red out: developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue.

As a pivotal signalling molecule involved in various physiological and pathological processes, nitric oxide (NO) has motivated increasing interest in the last few decades. Although a considerable number of fluorescent probes have been developed for NO imaging, the in situ tracking of this gas molecule in biological events remains a big challenge, mainly because of the relatively short excitation and/or emission wavelengths, which are subject to background interference and lowered collection efficiency in deep-tissue imaging. Herein, we report a far-red emissive (650 nm) two-photon (TP) excitable NRNO probe, using Nile Red as the TP fluorophore, for NO detection and imaging both in vitro and in vivo. The NRNO probe shows a fast (within 180 s) and specific fluorescence response toward NO with a limit of detection (LOD) as low as 46 nM. The excellent properties of NRNO enable it to sensitively detect both exogenously and endogenously generated NO in living cells. The "NIR in" and "far-red out" lights lead to improved penetrating ability, thus endowing the probe with high resolution for the illumination of deep tissues. It is therefore able to visualize the NO generation in a lipopolysaccharide (LPS)-mediated inflammation process for the first time. Our results demonstrate that NRNO could be a practical tool for studying the NO-related biological events. Moreover, this study also suggests the possibility of using Nile Red and its derivatives to develop far-red emissive TP probes, which is an important, yet undeveloped area.

A fast responsive two-photon fluorescent probe for imaging H2O2 in lysosomes with a large turn-on fluorescence signal

Hydrogen peroxide (H2O2) plays a crucial role in many biological processes in the human body. As our understanding of the complexity of physiological H2O2 in lysosome, investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed a new example of a fast responsive and lysosome-targeted two-photon H2O2 fluorescent probe (Lyso-HP) with a large turn-on fluorescence signal (80-fold fluorescence enhancement). The addition of H2O2 to Lyso-HP results a dramatic fluorescence enhancement around 550nm. The probe could image exogenous and endogenous H2O2 in living cells and the probe was located in lysosomes with high colocalization coefficient (0.96) compared with LysoTracker. The large fluorescence enhancement of the two-photon probe Lyso-HP renders it attractive for imaging H2O2 in living tissues with deep tissue penetration. Significantly, the probe is feasible for fluorescently monitoring H2O2 level changes in lysosomes and suitable for fluorescence imaging of H2O2 in living tissues with deep penetration by using two-photon microscopy.

Measurement of the Nucleus Area and Nucleus/Cytoplasm and Mitochondria/Nucleus Ratios in Human Colon Tissues by Dual-Colour Two-Photon Microscopy Imaging.

We developed two-photon (TP) probes for DNA (ABI-Nu), cytoplasm (Pyr-CT), and mitochondria (BF-MT). We found that ABI-Nu binds to AT in the minor groove, while ABI-Nu and BF-MT are effective for tracking in the cytoplasm and mitochondria, respectively. These probes showed very large effective two-photon action cross section values of 2230, 1555, and 790 Göppert-Mayer units (1 GM = 10−50 cm4 s photon−1molecule−1) at 740 nm with emission maxima at 473, 561, and 560 nm, respectively, in each organelle. Using these probes, we quantitatively estimated the mean nuclear area and the ratios of nuclei to cytoplasm and mitochondria to nuclei in human colon tissues by dual-colour two-photon microscopy imaging within 2 h after biopsy. The mean nuclear area and the nuclei to cytoplasm and mitochondria to cytoplasm ratios increased in the following order: normal colon mucosa <colon adenoma <colon adenocarcinoma. Furthermore, the nuclear areas of these tissues showed significant differences that were well outside of the ranges of experimental errors, indicating the diagnostic potential of this method.