Two-photon Probe Research Articles

Precise Labeling and Tracking of Lipid Droplets in Adipocytes Using a Luminescent ZnSalen Complex.

Designing two-photon probes for precise labeling of lipid droplets (LDs) and monitoring LD dynamics in adipocytes is of great significance to understand LD homeostasis. We herein report that a luminescent metal complex LD-TPZn can specifically image LDs in adipose cells and tissue using one- or two-photon fluorescence microscopy. Importantly, LD-TPZn exhibited higher specificity to LDs than the commercial dyes Nile Red and Bodipy 493/503, probably due to different cellular uptake pathways, that is, clathrin-mediated endocytosis and non-selectively passive diffusion, respectively. More importantly, LD-TPZn can be applied as a two-photon LD probe to image adipose tissue, one of the most challengeable tissues for traditional one-photon fluorescence microscope imaging due to the strong light scattering. Most importantly, LD-TPZn can be used to monitor LD growth during adipogenesis of preadipocytes, which is highly desirable to unravel the relationship between LD homeostasis and metabolic diseases.

Development of a Silicon-Rhodamine Based Near-Infrared Emissive Two-Photon Fluorescent Probe for Nitric Oxide.

Two-photon (TP) fluorescent probes are potential candidates for near-infrared (NIR) imaging which holds great promise in biological research. However, currently, most TP probes emit at wavelength <600 nm, which impedes their practical applications. In this work, we explored the TP properties of a silicon-rhodamine (SiR) derivative and hence developed the first SiR scaffold based "NIR-to-NIR" TP probe (SiRNO) for nitric oxide (NO). SiRNO exhibited high sensitivity and specificity, as well as fast response for NO detection. It was able to track the subtle variation of intracellular NO content in live cells. Owing to the NIR excitation and emission, SiRNO enabled the detection of NO in situ in the xenograft tumor mouse model, revealing the NO generation during the tumor progression. This work indicates that SiR can be an ideal platform for the development of NIR emissive TP probe and may thus promote the advancement of NIR imaging.

Two-Photon Probes for pH: Detection of Human Colon Cancer using Two-Photon Microscopy.

We have developed two-photon (TP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10-50cm4s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/3) solution. The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the TPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger Pacid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating much brighter TPM images and 6-fold larger Pacid values compared to normal tissue. These results suggest the potential utility of BHEt-1 for detecting colon cancer in human tissues using TPM.

Three-input “AND-Type” fluorescent logic gate as ratio probe for specific imaging of hypochlorite in rough endoplasmic reticulum

Optical ratio two-photon probe utilizing fluorescent responses for the imaging and detecting of hypochlorite (ClO−) require smart molecular systems able to operate selectively in the weak alkaline watery environment. Herein, we report a three-input “AND-type” fluorescent logic gate (NPA-CN) that demonstrates effective quantification of ClO− in the weak alkaline watery environment. NPA-CN with high signal-to-noise ratio provides a linear response to the three-input (ClO−, OH− and H2O) in rough endoplasmic reticulum (RER) of living cells on ratio-mode. More inportantly, NPA-CN can be used to whilst simultaneously image and quantify the changes of three-input.

Mitochondria-targeted iridium (III) complexes as two-photon fluorogenic probes of cysteine/homocysteine

Fluorescent imaging of cysteine (Cys) and homocysteine (Hcy) is crucial to understand sulphur metabolism in living cells. Whereas two-photon active cyclometalated Ir(III) complex for detecting Cys/Hcy has been rarely reported. Herein, two novel cyclometalated iridium (III) complexes Ir1-Ir2 with different electron-donor were designed, synthesized and fully characterized as well as confirmed by single-crystal X-ray diffraction analysis, which could selectively react with Cys/Hcy, accompanying with an obvious one- and two-photon fluorescence enhancement in vitro. The mechanism for sensing Cys/Hcy was further analyzed in detail by mass spectra and time-dependent density functional theory calculations. It was found that Ir2, bearing ester group, exhibited higher performance on the selective detection of Cys and Hcy compared to Ir1. Notably, the two-photon action cross-section value in the near-infrared region (∼800nm) of Ir2 is significantly enhanced upon the addition of Cys/Hcy. Further application to cellular imaging indicated that Ir2 is membrane permeable and capable of monitoring the changes of intracellular mitochondrial Cys/Hcy. These results support that such two-photon active complexes might provide a promising platform for the detection of mitochondrial Cys/Hcy in living biological systems.

Mitochondria-targeted two-photon fluorescent probe for the detection of biothiols in living cells

Mitochondrial thiols play a key role in maintaining the redox environment. In this work, three mitochondria-targeted two-photon probes (1–3) have been explored to detect biothiols in vitro and in vivo with a large turn-on fluorescence signal. These fluorescence probes display sensitive and selective response to thiols in the water environment, feature good linearity ranges with low detection limit. The negligible cytotoxicity, excellent photophysical properties and high selectivity of these probes will find their useful applications in biomedical research.

Efficient Two-Photon Fluorescent Probe for Glutathione S-Transferase Detection and Imaging in Drug-Induced Liver Injury Sample.

Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe P-GST for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2,4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, P-GST was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI.

A ratiometric two-photon probe for Ca2+ in live tissues and its application to spinal cord injury model.

Ratiometric imaging with a small-molecule probe is important for the in-situ quantitative analysis of chemical events. We developed a ratiometric two-photon fluorescent probe (SCa1-IREF) derived from dual dyes with different Stokes shifts. This probe has two identical windows: a Ca2+-sensing window and an internal reference window, with eliminated FRET interference. SCa1-IREF shows a marked change in the ratio upon response with Ca2+, significant two-photon brightness, considerable selectivity for Ca2+, and cell loading ability with low cytotoxicity. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca2+ in live neurons and various tissues including rat spinal cord tissue. The studies of spinal cord injury model revealed that the Ca2+ level was significantly affected by elapsed time after injury. These results will provide useful applications for in-situ [Ca2+]i imaging and for the development of effective ratiometric probes.

A highly selective two-photon probe with large turn-on signal for imaging endogenous HOCl in living cells

Hypochlorous acid (HOCl) is one of the typical reactive oxygen species (ROS), which plays a crucial role in the immune system, and is involved in many neurodegenerative disorders, such as ischemic stroke, Alzheimer's, and Parkinson's disease. A two-photon probe for sensing HOCl with high selectivity, fast response (within 30 s), good penetration depth and low cytotoxicity is reported. Under two-photon excitation, the probe exhibited a remarkable fluorescence ‘‘turn-on’’ response to HOCl. Furthermore, the probe was successfully applied to the monitoring of endogenous HOCl stimulated by lipopolysaccharide in living microglia BV-2 cells.

Construction of an alkaline phosphatase-specific two-photon probe and its imaging application in living cells and tissues.

Alkaline phosphatase (ALP) is a family of enzymes involved in the regulation of important biological processes such as cell differentiation and bone mineralization. Monitoring the activity of ALP in serum can help diagnose a variety of diseases including bone and liver diseases. There has been growing interest in developing new chemical tools for monitoring ALP activity in living systems. Such tools will help further delineate the roles of ALP in biological and pathological processes. Previously reported fluorescent probes has a number of disadvantages that limit their application, such as poor selectivity and short-wavelength excitation. In this work, we report a new two-photon fluorescent probe (TP-Phos) to selectively detect ALP activity. The probe is composed of a two-photon fluorophore, a phosphate recognition moiety, and a self-cleavable adaptor. It offers a number of advantages over previously reported probes, such as fast reaction kinetics, high sensitivity and low cytotoxicity. Experimental results also showed that TP-Phos displayed improved selectivity over DIFMUP, a commonly utilized ALP probe. The selectivity is attributed to the utilization of an ortho-functionalised phenyl phosphate group, which increases the steric hindrance of the probe and the active site of phosphatases. Moreover, the two-photon nature of the probe confers enhanced imaging properties such as increased penetration depth and lower tissue autofluorescence. TP-Phos was successfully used to image the endogenous ALP activity of hippocampus, kidney and liver tissues from rat.

An OFF-ON Two-Photon Fluorescent Probe for Tracking Cell Senescence in Vivo.

A naphthalimide-based two-photon probe (AHGa) for the detection of cell senescence is designed. The probe contains a naphthalimide core, an l-histidine methyl ester linker, and an acetylated galactose bonded to one of the aromatic nitrogen atoms of the l-histidine through a hydrolyzable N-glycosidic bond. Probe AHGa is transformed into AH in senescent cells resulting in an enhanced fluorescent emission intensity. In vivo detection of senescence is validated in mice bearing tumor xenografts treated with senescence-inducing chemotherapy.

A fast-responsive two-photon fluorescent turn-on probe for nitroreductase and its bioimaging application in living tissues

Hypoxia may improve the biological activity of nitroreductase (NTR). The development of effective methods for monitoring the activity of NTR plays an important role in the early diagnosis of tumors. Toward this goal, we have developed a new two-photon fluorescence NTR probe CoNO2-HY with fast response, high sensitivity and selectivity, and a large turn-on fluorescence signal. The probe CoNO2-HY could monitor endogenous NTR in living HeLa cells. Importantly, the probe CoNO2-HY is successfully applied to detect the endogenous NTR in the tumor tissue of mice with the penetration depth up to 90μm.

2-benzothiazoleacetonitrile based two-photon fluorescent probe for hydrazine and its bio-imaging and environmental applications

A novel turn-on two-photon fluorescent probe NS-N2H4 was developed with the 2-benzothiazoleacetonitrile as a new recognition site for the detection of hydrazine (N2H4). The two-photon probe exhibited favorable properties including high selectivity, low cytotoxicity and almost 16-fold fluorescence enhancement in the presence of N2H4 in solution. The probe could be used to image hydrazine in the living cells. Notably, we also used the two-photon fluorescent probe to image hydrazine in the tissue imaging for the first time. Furthermore, by the way of probe-loaded TLC plate, we further monitored vapor of hydrazine. Therefore, the novel two-photon probe is expected to be employed to detect N2H4 in biosamples and environmental pollution and the new recognition site will be widely applied to construct fluorescent probes for the detection of N2H4.

A mitochondrial-targeted two-photon fluorescent probe for imaging hydrogen sulfide in the living cells and mouse liver tissues

Hydrogen sulfide (H2S), as signaling molecule, plays a crucial role in many biological processes in living organisms. At the organelle level, we need to study tools to explain the complexity of physiological H2S in mitochondria. Toward this goal, we have developed a new example of a fast responsive and mitochondrial-targeted two-photon fluorescent H2S probe (MT-HS) with a large turn-on fluorescence signal (40-fold fluorescence enhancement). The addition of H2S to MT-HS results a dramatic fluorescence enhancement around 540nm. The probe could image exogenous and endogenous H2S in living cells and the probe was located in mitochondria with high colocalization coefficient compared with Mitochondrial-Tracker. The large fluorescence enhancement of the two-photon probe MT-HS renders it attractive for imaging H2S in living tissues with deep tissue penetration.

Two-photon fluorescence sensors for imaging NMDA receptors and monitoring release of Zn2+ from the presynaptic terminal

Synaptic Zn2+ plays an important role in neurotransmission and a neuromodulator. The development of the imaging tools for monitoring spatiotemporal changes taking place in synaptic Zn2+ concentrations is necessary in order to understand the role of Zn2+ in the function of many aspects of the glutamate system. In this work, two-photon probes 1 and 2, bearing ifenprodil-like tails that have affinity for NMDA receptors of neuronal cells, were designed and prepared. The two-photon fluorescent probe 1, which bears (N-(6-acetylnaphthalen-2-yl)-N-methylglycine) as two-photon fluorophore, enables high resolution imaging of neuronal cells. The two-photon fluorescent probe 2, which contains the di-2-picolylamine (DPA) as a Zn2+-binding site, the naphthalimide unit as the two-photon fluorophore, and the ifenprodil-like tail as the NMDA receptor binding moiety, can be employed for selective detection of Zn2+ located near the NMDA receptor and for monitoring concentration changes of Zn2+ in live neurons and hippocampal tissues.

A two-photon fluorescent probe for imaging aqueous fluoride ions in living cells and tissues

Fluoride ions are extensively found in aqueous solution, body fluids and tissues, and play an important function in life, so the detection and stable imaging of fluoride ions in living organs is of considerable importance. Herein, we designed and synthesized a new two-photon probe TXS to detect fluoride ions in aqueous solution with turn-on fluorescence. The chemical probe was constructed from tetrahydroxanthenone with a SiO bond to provide excellent selectivity for fluoride ions. Based on the emission properties of tetrahydroxanthenone, two-photon microscopy (TPM) was applied to image fluoride ions in the organs, and the results showed good images in liver tissue at a depth of 60 μm. TPM images of TXS supported a feasible application in biological analyses.

A two-photon fluorescent probe for real-time monitoring of autophagy by ultrasensitive detection of the change in lysosomal polarity.

The first two-photon probe, Lyso-OC, was proposed for use in monitoring cell autophagy by detection of the change in the lysosomal polarity during the membrane fusion process of autophagy. The Lyso-OC probe exhibited desirable optical properties and a detection signal that was selective to the polarity change. More importantly, Lyso-OC displayed real-time monitoring of autophagy in living cells.

A reversible ratiometric two-photon lysosome-targeted probe for real-time monitoring of pH changes in living cells.

Lysosome pH is known to be acidic (4.5-5.5) and has emerged as a critical physiological factor for lysosome activities and functions. Herein, we designed a novel ratiometric lysosome-targeted fluorescence resonance energy transfer (FRET) pH probe, which was fabricated by integrating the coumarin moiety (donor) with the naphthalimide moiety (acceptor). The sensing mechanism was essentially an integration of ICT and FRET processes, leading to the simultaneous intensity enhancement of coumarin and naphthalimide with a pH increase. Furthermore, morpholine was introduced as a lysosome-targeted group. Moreover, the probe could respond to pH in a proportional relationship at very broad range from pH 4.5 to 11.0 and showed remarkable advantages, including rapid response, high sensitivity and selectivity, suitable pKa of 5.62, and good reversibility. Furthermore, the probe was successfully used as a ratiometric TP lysosome-targeted fluorescence probe, not only for imaging of lysosomal pH, but also for visualizing chloroquine-induced changes of intracellular pH in real time in living cells with low cytotoxicity and autofluorescence. These proof-of concept studies demonstrate the practical application of the probe in biological systems.

A water-soluble two-photon ratiometric triarylboron probe with nucleolar targeting by preferential RNA binding.

By functionalizing triarylboron with cyclen, we developed a two-photon fluorescence probe, TAB-2, which can selectively bind RNA with a ratiometric readout. We tested TAB-2 in NIH/3T3 fibroblast cells, and demonstrated its capability in visualizing nucleoli and analyzing microenvironment polarity by two-photon and fluorescence-lifetime imaging microscopy.

A two-photon ratiometric ESIPT probe for fast detection and bioimaging of palladium species

A novel ratiometric ESIPT fluorescent probe, which is specific for palladium species of all the typical oxidation states (0, +2, and +4), has been designed. Notably, based on the excited state intramolecular proton transfer (ESIPT) process, the probe exhibits a ratiometric fluorescent response to palladium species with a low detection limit (9.0 nM, 0.96 ppb) in about 1.5 min at room temperature. Moreover, it has been successfully used as a two-photon ratiometric fluorescent palladium probe for in vivo and three-dimensional imaging with low cytotoxicity and autofluorescence. Compared with other reported palladium probes, the probe displays a shorter ratiometric response time and lower detection limit in milder test conditions. All of the results indicate that the probe may be favorable for environmental and biological applications.