Two-photon Probe Research Articles

Fluorinated triphenylamine phthalocyanine @ silica-coated gold nanorods: A photoactivated lysosome escape and targeting mitochondria two-photon probe for imaging-guided photothermal synergistic photodynamic therapy in cancer cells

The timely evasion of nanomedicines from lysosomes is essential to avert premature degradation under the acidic and hydrolytic conditions characteristic of these cellular compartments. However, the development of effective strategies has been hindered by the complexity of design material and the scarcity of practical methods. In this study, we have synthesized a novel nanoparticle, designated as TPA-BPAF-SiPc@AuNR@SiO2. This nanoparticle was prepared by encapsulating near-infrared fluorinated triphenylamine-substituted silicon phthalocyanines (TPA-BPAF-SiPc) within mesoporous silica-coated gold nanorods (AuNR@SiO2). TPA-BPAF-SiPc@AuNR@SiO2 functions as a dual-function two-photon probe, facilitating photoactivated lysosome escape and targeting mitochondria. The inherent aggregation-induced emission (AIE) two-photon fluorescence of TPA-BPAF-SiPc is notably bright when encapsulated in AuNR@SiO2 nanocarriers, a phenomenon not observed in polymer nanocarriers composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) or in THF/water mixtures. Upon irradiation, this nanoparticle autonomously escapes from lysosomes and selectively targets mitochondria, a process can be visually monitored in real-time through the two-photon AIE fluorescence of TPA-BPAF-SiPc. Moreover, upon activation, TPA-BPAF-SiPc@AuNR@SiO2 produces a substantial quantity of reactive oxygen species (ROS) and induces hyperthermia effects, showcasing its potential for effective photodynamic therapy (PDT) in conjunction with synergistic hyperthermia. Flow cytometry data corroborate the induction of tumor cell death through both necrosis and apoptosis pathways by TPA-BPAF-SiPc@AuNR@SiO2. This study underscores the potential of TPA-BPAF-SiPc@AuNR@SiO2 as a multifunctional probe capable of enabling lysosome escape, mitochondria targeting, and two-photon fluorescence imaging-guided photothermal synergistic photodynamic therapy, specifically tailored for the treatment of breast cancer.

Dual-responsive two-photon probe for specific lipid droplets near-infrared fluorescence imaging in the brain of epileptic mice

Abnormal lipid metabolism in glial cells is a key pathological feature of epilepsy. The identification of lipid droplets (LDs) is essential for investigating lipid metabolism, disease progression, and potential therapeutic interventions. Two-photon imaging technology enables real-time visualization of the spatial distribution and temporal dynamics of LDs in epilepsy models. In this study, we developed a novel two-photon excited dual-responsive near-infrared fluorescent probe, CabA, based on viscosity and polarity, to monitor dynamic changes in LDs. The fluorescence of CabA at 670 nm exhibits a significant increase in response to low polarity and high viscosity due to the twisted intramolecular charge transfer and intramolecular charge transfer mechanisms. The LDs-targeting capability of CabA at the cellular level and the process of LDs generation between neurons and astrocytes during the pathological advancement of epilepsy have been validated. In situ synchronous imaging experiments in epileptic and normal mice using CabA revealed abnormal LDs accumulation in the brain during seizures. Two-photon fluorescence imaging further demonstrated LDs accumulation in the brains of epileptic mice at a penetration depth of 100 μm. This study offers a valuable tool for enhancing the understanding of LDs in physiological and pathological processes, potentially aiding in the early diagnosis of epilepsy.

Non-adiabatic electronic relaxation of tetracene from its brightest singlet excited state.

The ultrafast relaxation dynamics of tetracene following UV excitation to the bright singlet state S6 has been studied with time-resolved photoelectron spectroscopy. With the help of high-level abinitio multireference perturbation theory calculations, we assign photoelectron signals to intermediate dark electronic states S3, S4, and S5 as well as to a low-lying electronic state S2. The energetic structure of these dark states has not been determined experimentally previously. The time-dependent photoelectron yields assigned to the states S6, S5, and S4 have been analyzed and reveal the depopulation of S6 within 60fs, while S5 and S4 are populated with delays of about 50 and 80fs. The dynamics of the lower-lying states S3 and S2 seem to agree with a delayed population coinciding with the depopulation of the higher-lying states S4-S6 but could not be elucidated in full detail due to the low signal levels of the corresponding two-photon ionization probe processes.

Promising Molecular Architectures for Two-Photon Probes in the Diagnosis of α-Synuclein Aggregates.

The abnormal deposition of protein in the brain is the central factor in neurodegenerative disorders (NDs). These detrimental aggregates, stemming from the misfolding and subsequent irregular aggregation of α-synuclein protein, are primarily accountable for conditions such as Parkinson's disease, Alzheimer's disease, and dementia. Two-photon-excited (TPE) probes are a promising tool for the early-stage diagnosis of these pathologies as they provide accurate spatial resolution, minimal intrusion, and the ability for prolonged observation. To identify compounds with the potential to function as diagnostic probes using two-photon techniques, we explore three distinct categories of compounds: Hydroxyl azobenzene (AZO-OH); Dicyano-vinyl bithiophene (DCVBT); and Tetra-amino phthalocyanine (PcZnNH2). The molecules were structurally and optically characterized using a multi-technique approach via UV-vis absorption, Raman spectroscopy, three-dimensional fluorescence mapping (PLE), time-resolved photoluminescence (TRPL), and pump and probe measurements. Furthermore, quantum chemical and molecular docking calculations were performed to provide insights into the photophysical properties of the compounds as well as to assess their affinity with the α-synuclein protein. This innovative approach seeks to enhance the accuracy of in vivo probing, contributing to early Parkinson's disease (PD) detection and ultimately allowing for targeted intervention strategies.

Two-Photon Absorption Aggregation-Induced Emission Luminogen/Paclitaxel Nanoparticles for Cancer Theranostics.

Multifaceted nanoplatforms integrating fluorescence imaging and chemotherapy have garnered acknowledgment for their potential potency in cancer diagnosis and simultaneous in situ therapy. However, some drawbacks remain for traditional organic photosensitizers, such as poor photostability, short excitation wavelength, and shallow penetration depth, which will greatly lower the chemotherapy treatment efficiency. Herein, we present lipid-encapsulated two-photon active aggregation-induced emission (AIE) luminogen and paclitaxel (PTX) nanoparticles (AIE@PTX NPs) with bright red fluorescence emission, excellent photostability, and good biocompatibility. The AIE@PTX NPs exhibit dual functionality as two-photon probes for visualizing blood vessels and tumor structures, achieving penetration depth up to 186 and 120 μm, respectively. Furthermore, the tumor growth of the HeLa-xenograft model can be effectively prohibited after the fluorescence imaging-guided and PTX-induced chemotherapy, which shows great potential in the clinical application of two-photon cell and tumor fluorescence imaging and cancer treatment.

Two-Photon Nanoparticle Probe for Formaldehyde Detection via the AILE Luminescence Mechanism.

A two-photon nanoparticle probe was designed and developed based on the principle of intermolecular interaction of the aggregation-induced locally excited emission luminescence mechanism. The probe has the advantages of simple synthesis, convenient use, strong atomic economy, good biocompatibility, and photobleaching resistance. It can produce a specific and sensitive response to formaldehyde, help detect FA in normal cells and cancer cells, and is expected to become a specific detection probe for FA in vitro and in vivo.

An antagonist-based two-photon fluorogenic probe for imaging metabotropic glutamate receptor 5 in neuronal cells

The expression of metabotropic glutamate receptor 5 (mGluR5) is subject to developmental regulation and undergoes significant changes in neuropsychiatric disorders and diseases. Visualizing mGluR5 by fluorescence imaging is a highly desired innovative technology for biomedical applications. Nevertheless, there are substantial problems with the chemical probes that are presently accessible. In this study, we have successfully developed a two-photon fluorogenic probe, mGlu-5-TP, based on the structure of mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP). Due to this antagonist-based probe selectively recognizes mGluR5, high expression of mGluR5 on living SH-SY5Y human neuroblastoma cells has been detected during intracellular inflammation triggered by lipopolysaccharides (LPS). Of particular significance, the probe can be employed along with two-photon fluorescence microscopy to enable real-time visualization of the mGluR5 in Aβ fiber-treated neuronal cells, thereby establishing a connection to the progression of Alzheimer's disease (AD). These results revealed that the probe can be a valuable imaging tool for studying mGluR5-related diseases in the nervous system.

Cascading Detection of Hydrogen Sulfide and N-Acetyltransferase 2 in Hepatocellular Carcinoma Cells Using a Two-Photon Fluorescent Probe.

Hydrogen sulfide (H2S), a critical gas signaling molecule, and N-acetyltransferase 2 (NAT2), a key enzyme in drug metabolism, are both known active biomarkers for liver function. However, the interactions and effects of H2S and NAT2 in living cells or lesion sites remain unknown due to the lack of imaging tools to achieve simultaneous detection of these two substances, making it challenging to implement real-time imaging and precise tracking. Herein, we report an activity-based two-photon fluorescent probe, TPSP-1, for the cascade detection of H2S and NAT2 in living liver cells. Continuous conversion from TPSP-1 to TPSP-3 was achieved in liver cells and tissues. Significantly, leveraging the outstanding optical properties of this two-photon fluorescent probe, TPSP-1, has been effectively used to identify pathological tissue samples directly from clinical liver cancer patients. This work provides us with this novel sensing and two-photon imaging probe, which can be used as a powerful tool to study the physiological functions of H2S and NAT2 and will help facilitate rapid and accurate diagnosis and therapeutic evaluation of hepatocellular carcinoma.

Chemical Strategies for the Detection and Elimination of Senescent Cells.

Cellular senescence can be defined as an irreversible stopping of cell proliferation that arises in response to various stress signals. Cellular senescence is involved in diverse physiological and pathological processes in different tissues, exerting effects on processes as differentiated as embryogenesis, tissue repair and remodeling, cancer, aging, and tissue fibrosis. In addition, the development of some pathologies, aging, cancer, and other age-related diseases has been related to senescent cell accumulation. Due to the complexity of the senescence phenotype, targeting senescent cells is not trivial, is challenging, and is especially relevant for in vivo detection in age-related diseases and tissue samples. Despite the elimination of senescent cells (senolysis) using specific drugs (senolytics) that have been shown to be effective in numerous preclinical disease models, the clinical translation is still limited due to the off-target effects of current senolytics and associated toxicities. Therefore, the development of new chemical strategies aimed at detecting and eliminating senescent cells for the prevention and selective treatment of senescence-associated diseases is of great interest. Such strategies not only will contribute to a deeper understanding of this rapidly evolving field but also will delineate and inspire new possibilities for future research.In this Account, we report our recent research in the development of new chemical approaches for the detection and elimination of senescent cells based on new probes, nanoparticles, and prodrugs. The designed systems take advantage of the over-representation in senescent cells of certain biomarkers such as β-galactosidase and lipofuscin. One- and two-photon probes, for higher tissue penetration, have been developed. Moreover, we also present a renal clearable fluorogenic probe for the in vivo detection of the β-galactosidase activity, allowing for correlation with the senescent burden in living animals. Moreover, as an alternative to molecular-based probes, we also developed nanoparticles for senescence detection. Besides, we describe advances in new therapeutic agents to selectively eradicate senescent cells using β-galactosidase activity-sensitive gated nanoparticles loaded with cytotoxic or senolytic agents or new prodrugs aiming to increase the selectivity and reduction of off-target toxicities of current drugs. Moreover, new advances therapies have been applied in vitro and in vivo. Studies with the probes, nanoparticles, and prodrugs have been applied in several in vitro and in vivo models of cancer, fibrosis, aging, and drug-induced cardiotoxicity in which senescence plays an important role. We discuss the benefits of these chemical strategies toward the development of more specific and sophisticated probes, nanoparticles, and prodrugs targeting senescent cells.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels

SignificanceNitric oxide (NO) is involved in the regulation of physiological and pathological mechanisms of the cardiovascular, nervous and immune systems; and plays an important role in cancer being involved in tumor growth and suppressing processes depending on its concentration. ApproachThe development of a near-infrared excitable nanoprobe, consisting of gold nanoparticles functionalized with a two-photon excitable NO probe, for the detection of intracellular NO is reported. ResultsThe nanoprobe showed good selectivity towards NO over cellular interferences and excellent stability in aqueous-medium over time. The nanoprobe was able to selectively detect endogenous and exogenous NO in different cell lines and it accumulated in the acidic organelles showing negligible toxicity. Importantly, the nanoprobe showed potential to quantify intracellular NO concentrations in breast cancer cells. ConclusionsThe novel gold-based two-photon nanoprobe showed an excellent performance and versatility and could potentially be applied for the spatiotemporal monitoring of in vivo NO levels.

Tumor Microenvironment-Regulating Two-Photon Probe Based on Bimetallic Post-Coordinated MOF Facilitating the Dual-Modal and Deep Imaging-Guided Synergistic Therapies.

The intricate tumor microenvironment (TME) always brings about unsatisfactory therapeutic effects for treatments, although nanomedicines have been demonstrated to be highly beneficial for synergistic therapies to avoid the side effects caused by the complexity and heterogeneity of cancer. Developing nanotheranostics with the functionalities of both synergistic therapies and TME regulation is a good strategy but is still in its infancy. Herein, an "all-in-one" nanoplatform for integrated diagnosis and treatment, namely, Carrier@ICG@DOX@FA (CIDF), is constructed. Benefiting from the bimetallic coordination of Eu3+-HTHA (4,4,4-trifluoro-1-(9-hexylcarbazol-3-yl)-1,3-butanedione) and Fe3+ with the ligands in UiO-67, CIDF can simultaneously achieve two-photon fluorescence imaging, fluorescent lifetime imaging in deep tumors, and regulation of TME. Owing to its porosity, CIDF can encapsulate indocyanine green as photosensitizers and doxorubicin as chemotherapeutic agent, further realizing light-controlled drug release. Moreover, CIDF exhibited good biocompatibility and tumor targeting by coating with folic-acid-modified polymers. Both in vitro and in vivo experiments demonstrate the excellent therapeutic efficacy of CIDF through dual-modal-imaging-guided synergistic photothermal-, photodynamic-, and chemotherapy. CIDF provides a new paradigm for the construction of TME-regulated synergistic nanotheranostics and realizes the complete elimination of tumors without recurrence.

Real-Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases.

Intravital fluorescence imaging of functional osteoclasts within their intact disease context provides valuable insights into the intricate biology at the microscopic level, facilitating the development of therapeutic approaches for osteoclast-associated bone diseases. However, there is a lack of studies investigating osteoclast activity within deep-seated bone lesions using appropriate fluorescent probes, despite the advantages offered by the multi-photon excitation system in enhancing deep tissue imaging resolution. In this study, we report on the intravital tracking of osteoclast activity in three distinct murine bone disease models. We utilized a cathepsin K (CatK)-responsive two-photon fluorogenic probe (CatKP1), which exhibited a notable fluorescence turn-on response in the presence of active CatK. By utilizing CatKP1, we successfully monitored a significant increase in osteoclast activity in hindlimb long bones and its attenuation through pharmacological intervention without sacrificing mice. Thus, our findings highlight the efficacy of CatKP1 as a valuable tool for unraveling pathological osteoclast behavior and exploring novel therapeutic strategies.

Observation of formaldehyde-induced ER stress by an ER-targeting two-photon probe

The presence of formaldehyde (FA) residue in leather during processing impacts the quality of the leather. Additionally, FA penetrates the nervous system through inhalation and skin contact, resulting in neuroinflammation and the build-up of misfolded proteins in the brain. Furthermore, it disrupts the endoplasmic reticulum, leading to abnormal protein aggregation and endoplasmic reticulum stress. As a result, the development of a dependable approach for monitoring endoplasmic reticulum stress caused by FA in scrap leather is crucial. With this research we developed a two-photon fluorescence probe (SKD-1-HCHO) sensitive to FA and targeting the endoplasmic reticulum. The extent of endoplasmic reticulum stress during the neuroinflammation process induced by scrap leather was investigated. According to the experimental results, the probe displayed a rapid Turn-ON response, remarkable specificity, and high sensitivity toward FA in the two-photon mode. Moreover, the probe exhibited an extensive linear range both in vitro and in cells. The induction of neuroinflammation by scrap leather was observed through BV-2 cells and mice. It was found that the formaldehyde content in scrap leather could induce cellular inflammatory responses, leading to initiation of endoplasmic reticulum stress processes. Significantly, while the anti-inflammatory drug NS-398 could not regulate the formaldehyde levels in cells, it could significantly inhibit cellular inflammation and the process of endoplasmic reticulum stress.

Rational design of a two-photon probe with solvatochromic emission and AIE-activity for lipid droplet specific imaging

Abnormal changes in numbers, sizes and polarities of lipid droplet (LD) are thought to be closely associated with varieties diseases such as fatty liver, diabetes, atherosclerosis and even cancer. However, there still exists some shortcomings of the current commercial LD probes, such as aggregation-caused quenching (ACQ) effect, small Stokes shifts, excitation wavelength restricted to visible light region, and lacking of two-photon excitation fluorescence, which restrict the diagnosis of LD related diseases. Herein, a two-photon aggregation-induced emission (AIE) bioprobe, namely TP-LDP, was rationally designed by constructing of the donor-acceptor-donor (D-A-D) structure for specific imaging of LD. TP-LDP exhibited AIE-activities, solvatochromic emissions (λemmax: 451–652 nm), extra-large Stokes shifts (11550 cm−1) as well as two-photon absorption ability (156 GM). More importantly, cellular experiments demonstrated that TP-LDP possessed excellent photostability, nice two-photon excited fluorescence imaging ability as well as precise LD-targeting, and successfully detected abnormal LD proliferations in living cells. These features enable TP-LDP to effectively compensate for the shortcomings of commercial LD dyes and have the potential to become a powerful tool for detecting LD related diseases.

Two-photon fluorescent chemosensors based on the GFP-chromophore for the detection of Zn2+ in biological samples – From design to application

A library of twelve novel fluorescent GFP chromophore based chemosensors (GFZnPs) was prepared for the detection of Zn2+ in biological samples with two-photon microscopy. The presented compounds are the hybrids of the Zn2+ chelator 8-aminoquinoline motif and the GFP chromophore, exploiting and enhancing the properties of the natural chromophore. In the presented probes, the quenching rotation around the benzylidene carbon of the GFP chromophore is conditionally blocked upon Zn2+ binding, resulting in a fluorescent response to the ion. GFZnPs have an efficient and simple synthesis making them one of the most easily accessible two-photon (2P) Zn2+ sensors. We present the comprehensive spectroscopical characterization of the probe family highlighting their bright fluorescent emission (ε × Φ > 200) at λem∼520 nm upon excitation by λex∼450 nm light. The sensors feature high selectivity for Zn2+ with an affinity in the nanomolar range, excellent water solubility, and usability at a wide pH-range (pH > 6). The relationship between the structure of compounds and their properties was studied and rationalized by theoretical considerations. The 2P-action cross section of the probes reaches δ × Φcomp > 5 GM at λex,2P= 900 nm, with an increase up to 200-fold, which makes them one of the best two-photon probes for Zn2+. The applicability of selected probes was demonstrated by epifluorescent and 2P microscopy on cultured cells and brain slices. This work expands the toolbox of efficient two-photon zinc sensors with a valuable new family.

Rhenium diselenide nanosheets as an excellent bi-color probe for intracellular two-photon imaging

Recently two-dimensional (2D) nanomaterials-mediated photothermal therapy (PTT) has become an important tool for treating cancer. However, visually monitoring the localization of nanoagents in cancer cells remains a key challenge. Herein, exploring high photostability, and high biocompatibility probes for monitoring PTT are still highly desirable. In this study, a novel optical probe named PEG@ReSe2 nanosheets (polyethylene glycol, PEG; rhenium diselenide, ReSe2) was prepared and employed to perform in vivo two-photon imaging for monitoring PTT in esophageal cancer cells. Under the illumination of a near-infrared (NIR) femtosecond laser at 820 nm with the output power as low as 5 mW, the PEG@ReSe2 nanosheets showed significant two-photon features, producing a significant photoluminescence emission band at 637 nm. In addition, the prepared PEG@ReSe2 nanosheets exhibited high resistance to photobleaching. Also, the PEG@ReSe2 nanosheets showed low cytotoxicity for cancer cells, providing a high viability of more than 90%. More importantly, the PEG@ReSe2 nanosheets could enter cancer cells and work as a two-photon probe for visually displaying the PTT process. After 12 h incubation, the PEG@ReSe2 nanosheets produced strong two-photon signals in KYSE150 cancer cells, enabling them to perform in vivo bi-channel bioimaging. In light of its photothermal effects, the PEG@ReSe2 nanosheets also could serve as a sensor for monitoring PTT under low-power infrared laser irradiation. This study suggests that, the PEG@ReSe2 nanosheets could serve as a superior two-photon biosensor for monitoring the PTT process in cancer nanomedicine.

A general design of pyridinium-based fluorescent probes for enhancing two-photon microscopy

Two-photon absorbing fluorescent probes have emerged as powerful imaging tools for subcellular-level monitoring of biological substances and processes, offering advantages such as deep light penetration, minimal photodamage, low autofluorescence, and high spatial resolution. However, existing two-photon absorbing probes still face several limitations, such as small two-photon absorption cross-section, poor water solubility, low membrane permeability, and potentially high toxicity. Herein, we report three small-molecule probes, namely MSP-1arm, Lyso-2arm, and Mito-3arm, composed of a pyridinium center (electron-acceptor) and various methoxystyrene “arms” (electron-donor). These probes exhibit excellent fluorescence quantum yield and decent aqueous solubility. Leveraging the inherent intramolecular charge transfer and excitonic coupling effect, these complexes demonstrate excellent two-photon absorption in the near-infrared region. Notably, Lyso-2arm and Mito-3arm exhibit distinct targeting abilities for lysosomes and mitochondria, respectively. In two-photon microscopy experiments, Mito-3arm outperforms a commercial two-photon absorbing dye in 2D monolayer HeLa cells, delivering enhanced resolution, broader NIR light excitation window, and higher signal-to-noise ratio. Moreover, the two-photon bioimaging of 3D human forebrain organoids confirms the successful deep tissue imaging capabilities of both Lyso-2arm and Mito-3arm. Overall, this work presents a rational design strategy in developing competent two-photon-absorbing probes by varying the number of conjugated “arms” for bioimaging applications.

Overcome the "Buckets Effect": Integration of AIEgens into Proteins for Fluorescence-Enhanced Two-Photon Imaging.

Luminogens with aggregation-induced emission characteristics (AIEgens) are considered good options for two-photon (2P) probes, owing to their flexibility of design, heavy-metal-free composition, and resistance to photobleaching. However, the design principles for large 2P absorption cross-section (δ) generally require high coplanarity, strong donor-acceptor (D-A) interactions, and long conjugation, which can severely weaken the brightness of AIEgens at the aggregated state and undermine their potential in 2P fluorescence imaging (2PFI). Exploration of a feasible approach to overcome the "Buckets Effect" of AIEgen-based 2P probes is thus a fascinating yet challenging task. Herein, an AIEgen, namely (Z)-2-(4-aminophenyl)-3-(5-(4-(bis(4-methoxyphenyl)amino)phenyl)thiophen-2-yl)acrylonitrile (MTAA) is designed to have a big δ according to the calculation result and a low fluorescence quantum yield (QY) of 2.2% in dimethyl sulfoxide (DMSO). Impressively, upon integrating into bovine serum albumin (BSA), the protein-sized MTAA@BSA dots exhibit a 25-fold higher fluorescence QY compared to MTAA molecules, contributing to an imaging depth of 818µm in the brain vasculature. The retention and clearance of MTAA@BSA dots in the liver and kidney are also studied using 2PFI. Overall, this work provides a facile approach to overcome the "Buckets Effect" of AIEgen to generate highly efficient, reliable, and biocompatible 2P probes.

Ultrasensitive two-photon probes for rapid detection of β-galactosidase during fruit softening and cellular senescence

Senescence is happening in every corner of the living organisms. β-galactosidase (β-gal) is one of the most important biomarkers during senescence in both plant and mammalian cells. Most β-gal fluorescent probes were focused on bio-imaging, only a few probes were developed for the detection of β-gal in fruit, and the probes that could detect β-gal in both fruits and living cells were even less. Here, two β-gal probes (TNap-βGal and TBNap-βGal) were synthesized, which can not only image the increase of β-gal during both fruits softening and cellular senescence, but also prove that bananas are not suitable for storage in refrigerator and the subsequent accumulation of β-gal still in lysosome of mammalian cells. In addition, TNap-βGal was successfully applied to two-photon imaging of endogenous β-gal in both hDPMSCs and tissues of human dental pulp for the first time.

A two-photon mitochondria-targeting azo reductase probe for imaging in tumor cells and mice

Herein, a two-photon mitochondria-targeting fluorescent probe (TPNP) which can be activated by the azo reductase (AzoR) has been developed for imaging the hypoxic microenvironment of tumors in vivo. The probe TPNP showed a low detection limit (LOD = 1.85 eq), high selectivity, and considerable steadiness in different pH conditions (3.0–11.0). In living cells, it could co-locate with the commercial dye in the mitochondria (co-localization coefficient: 0.92) and indicate hypoxia in both one-photon and two-photon fluorescence imaging. Its AzoR-activation mechanism was further verified by the docking simulation as well as the experiments of adding AzoR or electron transporter inhibitors. Moreover, the in vivo real-time fluorescence imaging proved that TPNP could monitor hypoxia and the tumor progression in mouse tumor models. Overall, this designed probe exhibited high sensitivity, synthetic ease, and excellent fluorescent properties, which might therefore enlighten new ideas for the diagnosis of tumors in future studies.