Two-photon Fluorescent Probe Research Articles

Monitoring Glutathione Content of the Endoplasmic Reticulum under Scrap Leather-Induced Endoplasmic Reticulum Stress via an Endoplasmic Reticulum-Targeted Two-Photon Fluorescent Probe.

Maintaining tissue homeostasis necessitates the coordinated efforts of various cell types to regulate inflammation. Endoplasmic reticulum (ER) stress, a hallmark of inflammation, exacerbates tissue pathology in various human diseases. Glutathione (GSH), a pivotal regulator of cellular redox balance, controls disulfide bond formation in the ER, thereby shielding cells from oxidative stress. In this study, we developed a two-photon fluorescent probe, ER-GSH, with specific ER targeting and demonstrated its high sensitivity and rapid response to GSH. Experiments conducted on BV2 cells and a mice model of neuroinflammation induced by scrap leather revealed that inflammatory reactions led to ER stress and a substantial reduction in GSH levels. Notably, the anti-inflammatory drug NS-398 effectively inhibited cell inflammation and ER stress by maintaining GSH levels. These findings underscore the potential therapeutic significance of modulating GSH levels to alleviate the impact of neuroinflammation.

A highly specific two-photon fluorescent probe for real-time monitoring of acetylcholinesterase in neurogenic disorders in vivo

Acetylcholinesterase (AChE) hydrolyses choline into thiocholine, which is essential for cholinergic neurons to revert to their resting state following activation. Abnormal changes in AChE activity can directly affect nervous system function. Thus, the specific detection of AChE activity is urgently needed for elucidating the function of the nervous system and diagnosing AChE-related diseases. Current methods for detecting AChE activity have several limitations, including strong background interference and poor tissue penetration. Thus, we designed and synthesized a two-photon (TP) excited fluorescent probe, WZ-AChE, for the specific detection of AChE. Briefly, a carbamate bond was chosen to specifically recognize AChE, which can also be cleaved by AChE. The product, WZ, released strong deep red fluorescence signal under TP excitation at 800 nm. Our results showed that WZ-AChE can detect AChE activity in PC12 cells with both superior sensitivity and selectivity. In addition, we successfully applied WZ-AChE to a C. elegans Parkinson's disease (PD) model and a mouse model of depression. The findings revealed that AChE activity was greater in both disease models than in the control group. To summarize, a novel tool was created to investigate the mechanisms underlying PD and depression.

Excitation energy transfer based naphthalimide-boron-dipyrromethene dyads as two-photon fluorescent probes for palladium(0) detection and live cell imaging

Increasing industrial demand has accelerated the consumption and contamination of palladium (Pd) worldwide. The implementation of fluorescent probes, especially with two-photon excitation, holds great promise for Pd detection in environmental and biological analysis. In this work, two naphthalimide (NPI)-boron-dipyrromethene (BDP) dyads (named NBH and NBN) were developed as two-photon fluorescent probes for Pd(0) detection based on an excitation energy transfer (EET) strategy, employing NPI as the energy donor and BDP as the acceptor. Spectroscopic studies revealed that both probes exhibited a fast response to Pd(0) in a pronounced fluorescence "ON-OFF" manner. The Pd-catalyzed Tsuji-Trost reaction activated an intramolecular photoinduced electron transfer (PET) process between the aniline and BDP moieties, resulting in the fluorescence quenching of the BDP fluorophore. The reaction-based probes exhibited excellent sensitivity and selectivity for Pd(0) species with a detection limit as low as 2.4 nM. These probes were successfully applied to the determination of Pd residues in drug, soil, and water samples. A simple smartphone-based platform was also constructed to determine Pd(0) via an RGB reader. With negligible cytotoxicity, confocal imaging study validated their feasibility of monitoring Pd(0) in living cells through multiple excitation channels.

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.

Covalent assembly-based two-photon fluorescent probes for in situ visualizing nitroreductase activities: From cancer cells to human cancer tissues

Nitroreductase (NTR) is widely regarded as a biomarker whose enzymatic activity correlates with the degree of hypoxia in solid malignant tumors. Herein, we utilized 2-dimethylamino-7-hydroxynaphthalene as fluorophore linked diverse nitroaromatic groups to obtain four NTR-activatable two-photon fluorescent probes based on covalent assembly strategy. With the help of computer docking simulation and in vitro assay, the sulfonate-based probe XN3 was proved to be able to identify NTR activity with best performances in rapid response, outstanding specificity, and sensitivity in comparison with the other three probes. Furthermore, XN3 could detect the degree of hypoxia by monitoring NTR activity in kinds of cancer cells with remarkable signal-to-noise ratios. In cancer tissue sections of the breast and liver in mice, XN3 had the ability to differentiate between healthy and tumorous tissues, and possessed excellent fluorescence stability, high tissue penetration and low tissue autofluorescence. Finally, XN3 was successfully utilized for in situ visualizing NTR activities in human transverse colon and rectal cancer tissues, respectively. The findings suggested that XN3 could directly identify the boundary between cancer and normal tissues by monitoring NTR activities, which provides a new method for imaging diagnosis and intraoperative navigation of tumor tissue.

Efficient Near-Infrared Fluorophores Based on Cyanostyrene Derivatives for Two-Photon Fluorescence Bioimaging.

Organic fluorescent materials with red/near-infrared (NIR) emission are highly promising for use in biotechnology due to their exceptional advantages. However, traditional red/NIR fluorophores often exhibit weak emission at high concentrations or in an aggregated state due to the aggregate-caused quenching effect, which severely limits their applicability in biological imaging. To address this challenge, we developed a series of cyanostyrene derivatives with aggregation-induced emission characteristics, including 2,3-Bis-(4-styryl-phenyl)-but-2-enedinitrile (DPB), 2,3-Bis-{4-[2-(4-methoxy- phenyl)-vinyl]-phenyl}-but-2-enedinitrile (DOB), 2,3-Bis-{4-[2-(4-diphenylamino- phenyl)-vinyl]-phenyl}-but-2-enedinitrile (DTB), and 2,3-Bis-[4-(2-{4-[phenyl- (4-triphenylvinyl-phenyl)-amino]-phenyl}-vinyl)- phenyl]-but-2-enedinitrile (DTTB). Notably, these compounds exhibited intense solid state fluorescence owing to AIE effect, especially DTTB shows NIR emission with high solid state quantum efficiency (712 nm, ΦF=14.2 %). Then we prepared DTTB@PS-PEG NPs nanoparticles by encapsulating DTTB with the amphiphilic polymer polystyrene-polyethylene glycol (PS-PEG). Importantly, DTTB@PS-PEG NPs exhibited highly efficient NIR luminescence (ΦF=28.7 %) and a large two-photon absorption cross-section (1900 GM) under 800 nm laser excitation. The bright two-photon fluorescence of DTTB@PS-PEG indicated that it can be a highly promising candidate for two-photon fluorescence probe. Therefore, this work provides valuable insights for the design of highly efficient and NIR-emitting two-photon fluorescent probes.

A two-photon fluorescent probe sensitive to NADPH reveals metabolic aberrations in brain cells arising from mitochondrial dysfunction in Alzheimer’s disease

Mitochondrial dysfunction is implicated in the pathogenesis of numerous neurodegenerative disorders, including Alzheimer’s disease (AD). Nicotinamide adenine dinucleotide phosphate (NADPH) plays a crucial cofactor as a cofactor in diverse biological processes, with the interconversion between NADP+ and NADPH serving a pivotal function in intracellular reductive metabolism and redox homeostasis. Herein, we present a fast, ultrasensitive, two-photon fluorescent probe (NATP-3) with large Stokes shifts for real-time detection of NADPH levels in mitochondria of the brain cells and elucidate mitochondrial damage in AD. Significantly reduced levels of NAD(P)H in living brain cells under Aβ stress or in AD model animals such as C. elegans were intuitively observed, suggesting that cellular mitochondrial dysfunction and metabolic disorders in the brain are closely related to AD. Importantly, utilizing a high-throughput screening platform based on NATP-3, we identified apigenin as a promising candidate for mitigating mitochondrial dysfunction in AD. Notably, apigenin reinstates mitochondrial NAD(P)H production, thereby providing a potential entity molecule for intervening in mitochondrial damage during AD. This work provides a valuable tool for detecting and intervening in mitochondrial function and metabolic homeostasis associated with AD processes, emphasizing the importance of understanding the molecular mechanisms of mitochondrial dysfunction associated with neurodegenerative diseases.

Ratiometric two-photon fluorescent probe for imaging hydrogen peroxide during stroke-induced ferroptosis

Ischemic stroke (IS) initiates a programmed cell death through apoptosis, autophagy, and ferroptosis, which results in neuronal damage and brain injury. However, existing methods for in situ detection of ferroptosis are currently insufficient. Therefore, we constructed a series of hydrogen peroxide (H2O2—one of the major ROS) probes (QH2O2, MQH2O2, and BQH2O2) with quinoline derivatives as two-photon fluorophores and boronate as the recognition domain. Experimental results indicated that the linear response range of the probe MQH2O2 to H2O2 increased from < 10–300 μM as compared with the probe QH2O2. Moreover, the linear correlation coefficient also increased from 0.9012 to 0.999, indicating that the ratiometric fluorescent probe has built-in corrections for environmental interference, which allows a more accurate fluorescence analysis. Modification of the probe by connecting triethylene glycol monomethyl ether (BQH2O2) increased the lipophilic index of the probe to 2.01, which enhanced its ability to cross the blood–brain barrier. Therefore, the developed probe is useful for analyzing the H2O2 production during an ischemic stroke and its fluctuation with ferroptosis.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•−), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•− changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•− and HSCs targeting peptides. We observe that O2•− in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•−, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Multicolor Two-Photon Microscopy Imaging of Lipid Droplets and Liver Capsule Macrophages In Vivo.

Lipid droplets (LDs) store energy and supply fatty acids and cholesterol. LDs are a hallmark of chronic nonalcoholic fatty liver disease (NAFLD). Recently, studies have focused on the role of hepatic macrophages in NAFLD. Green fluorescent protein (GFP) is used for labeling the characteristic targets in bioimaging analysis. Cx3cr1-GFP mice are widely used in studying the liver macrophages such as the NAFLD model. Here, we have developed a tool for two-photon microscopic observation to study the interactions between LDs labeled with LD2 and liver capsule macrophages labeled with GFP in vivo. LD2, a small-molecule two-photon excitation fluorescent probe for LDs, exhibits deep-red (700 nm) fluorescence upon excitation at 880 nm, high cell staining ability and photostability, and low cytotoxicity. This probe can clearly observe LDs through two-photon microscopy (TPM) and enables the simultaneous imaging of GFP+ liver capsule macrophages (LCMs) in vivo in the liver capsule of Cx3cr1-GFP mice. In the NAFLD mouse model, Cx3cr1+ LCMs and LDs increased with the progress of fatty liver disease, and spatiotemporal changes in LCMs were observed through intravital 3D TPM images. LD2 will aid in studying the interactions and immunological roles of hepatic macrophages and LDs to better understand NAFLD.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE

BackgroundCysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. ResultsHere, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL). SignificanceThe outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

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.

A near-infrared fluorescent probe with two-photon excitation for in situ imaging of NQO1 in human colorectum cancer tissue

Colorectum cancer has become one of the most fatal cancer diseases, in which NAD(P)H: quinone oxidoreductase 1 (NQO1) plays a role in intracellular free radical reduction and detoxification and has been linked to colorectum cancer and chemotherapy resistance. Therefore, rational design of optical probe for NQO1 detection is urgent for the early diagnosis of colorectum cancer. Herein, we have developed a novel two-photon fluorescent probe, WHFD, which is capable of selectively detecting of intracellular NQO1 with two-photon (TP) absorption (800 nm) and near-infrared emission (620 nm). Combination with a substantial Stokes shift (175 nm) and biocompatibility, we have assessed its suitability for in vivo imaging of endogenous NQO1 activities from HepG2 tumor-bearing live animals with high tissue penetration up to 300 μm. Particularly, we for the first time used the probe to image NQO1 activities from human colorectum cancer samples by using TP microscopy, and proving our probe possesses reliable diagnostic performance to directly in situ imaging of cancer biomarker and can clearly distinguish the boundary between human colorectum cancer tissue and their surrounding normal tissue, which shows great potential for the intraoperative navigation.

Near-Infrared Light-Excited Quinolinium-Carbazole Small Molecule as Two-Photon Fluorescence Nucleic Acid Probe.

This article reports three new two-photon absorption (TPA) materials that are quinolinium-carbazole derivates. They are 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (M4), 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (H2), and 3-(N-methyl-4-ethylquinolinium iodide)-9-ethylcarbazole (H4). Their TPA cross-sections are 491, 515, and 512 GM, respectively. Under the excitation of near-infrared light, their fluorescence emission is about 650 nm. The compounds can stain nucleic acid DNA with the same level of nuclear localization as Hoechst 33342. Under continuous irradiation with a near-infrared laser, the three new compounds showed less fluorescence decay than DAPI, and the average fluorescence decay rates were 0.016%/s, 0.020%/s, and 0.023%/s. They are expected to become new two-photon fluorescent probes of nucleic acid DNA because of their excellent performance.

A highly specific two-photon fluorescent probe for pantetheinase and its biological applications

Pantetheinase is an enzyme that catalyzes the hydrolysis of pantetheine, releasing pantothenic acid (vitamin B5) and cysteamine as byproducts, which is associated with many physiological and pathological processes. In this work, a two-photon fluorescent probe PA-BAN for pantetheinase based on N-butyl-4-amino-1,8-naphthalimide fluorophore scaffold, was designed, synthesized, and developed for sensing the pantetheinase activity in various scenarios. In consideration of the catalytic characteristics and substrate preference of pantetheinase, we selected pantothenic acid (PA) as the recognition group and conjugated it to the fluorophore through amidation reaction. The fluorescence intensity of PA-BAN displayed an ideal linear correlation with the concentration of pantetheinase in vitro, with a detection limit of 0.18 ng/mL. PA-BAN also performed well for the visualizing of pantetheinase in live cells, zebrafish, and tissue slices. Moreover, given the pivotal role of pantetheinase in the host's resistance to malaria infection, PA-BAN was utilized to assess the serum pantetheinase activity of Plasmodium-infected mice. We observed a potential link between serum pantetheinase levels and infection severity, especially concerning cerebral malaria (CM) in the experiments of mice. Low serum pantetheinase levels potentially indicate high susceptibility to CM, making it serve as risk factor for individuals infected with malaria. Thus, the utility of PA-BAN in measuring serum pantetheinase levels could add to current diagnostic toolkit for malaria infection, contributing to a more comprehensive assessment of malaria infection.

Golgi-targeting viscosity probe for the diagnosis of Alzheimer’s disease

Early diagnosis and intervention of Alzheimer’s disease (AD) are particularly important to delay the pathological progression. Although fluorescent probes have been widely employed for investigating and diagnosing AD, their biological applications are significantly restricted due to the low penetration ability of the blood–brain barrier (BBB) in vivo. In this study, we reported the first Golgi-targeted two-photon (TP) fluorescent probe, DCM-DH, for detecting viscosity in the Golgi apparatus. The probe was rationally designed to exhibit superior analytical performance including high sensitivity, specific Golgi-targeting, efficient BBB penetration ability, and deep tissue penetration (247 μm) in the brains of AD model mice. Using the probe, we demonstrated that the fluorescence intensity in the human liver cancer cell (HepG2 cells) was higher than that of human normal liver cell (LO2 cells), and the brain viscosity of AD model mice increased significantly. We anticipate that this competent tool could be easily extended to other AD biomarkers for fundamental research on this detrimental disease.

Rational design of anthocyanidins-directed near-infrared two-photon fluorescent probes.

Recently, two-photon fluorescent probes based on anthocyanidin molecules have attracted extensive attention due to their outstanding photophysical properties. However, there are only a few two-photon excited fluorescent probes that really meet the requirements of relatively long emission wavelengths (>600 nm), large two-photon absorption (TPA) cross-sections (300 GM), significant Stokes shift (>80 nm), and high fluorescence intensity. Herein, the photophysical properties of a series of anthocyanidins with the same substituents but different fluorophore skeletons are investigated in detail. Compared with b-series molecules, a-series molecules with a six-membered ring in the backbone have a slightly higher reorganization energy. This results in more energy loss upon light excitation, enabling the reaction products to detect NTR through a larger Stokes shift. More importantly, there is very little decrease in fluorescence intensity as the Stokes shift increases. These features are extremely valuable for high-resolution NTR detection. In light of this, novel 2a-n (n = 1-5) compounds are designed, which are accomplished by inhibiting the twisted intramolecular charge transfer (TICT) effect through alkyl cyclization, azetidine ring and extending π conjugation. Among them, 2a-3 gains a long emission spectrum (λem = 691.4 nm), noticeable TPA cross-section (957 GM), and large Stokes shift (110 nm), indicating that it serves as a promising candidate for two-photon fluorescent dyes. It is hoped that this work will offer some insightful theoretical direction for the development of novel high performance anthocyanin fluorescent materials.

Visualization of peroxynitrite/GSH cross-talk in the oxidant-antioxidant balance by a dual-fluorophore and dual-site based mito-specific fluorescent probe.

Peroxynitrite (ONOO-) and glutathione (GSH) play mutually regulating roles in the oxidant-antioxidant balance of organisms, which has a profound relationship with people's health and disease. In this study, we designed a two-photon fluorescent probe CD-NA that could simultaneously detect ONOO- and GSH via dual-fluorophore and dual-site properties. CD-NA shows different fluorescence responses to ONOO- (annihilated red fluorescence) and GSH (enhanced green emission) with high specificity and sensitivity. Notably, the response of CD-NA to ONOO- was unaffected by GSH, and the reverse is also true. It allows the ONOO-/GSH cross-talk to be successfully imaged. Given these excellent properties, CD-NA has been favorably employed in detecting ONOO- and GSH in living cells with the ability to target mitochondria. Therefore, CD-NA offers an efficient method for understanding the oxidant-antioxidant balance and interrelated physiological functions of ONOO- and GSH in living systems, and provides a new strategy to sort out the complex relationships and roles of various analytes in complex physiological processes.

Excellent ratiometric two-photon fluorescent probes for hydrogen sulfide detection based on the fluorescence resonance energy transfer mechanism.

Fluorescence resonance energy transfer (FRET) is an important mechanism to design ratiometric fluorescent probes that are able to detect analytes quantitatively according to the ratio of two well-resolved emission signals. Two-photon (TP) fluorescent probes can realize the detection in living cells and tissues with deeper penetration depth, higher resolution, and lower photodamage in contrast to one-photon fluorescent probes. However, to date, fabricating TP-FRET ratiometric fluorescent probes possessing large two-photon absorption (TPA), high fluorescence quantum yield and perfect FRET efficiency is still challenging. Consequently, to develop excellent TP-FRET ratiometric probes and explore the relationship between their molecular structures and TP fluorescence properties, in this paper, we designed a series of H2S-detecting TP fluorescent probes employing the FRET mechanism based on an experimental probe BCD. Thereafter, we comprehensively evaluated the TP sensing performance of these probes by means of time-dependent density functional theory and quadratic response theory. Furthermore, we determined energy transfer efficiency and fluorescence quantum yield. Significantly, through regulating benzene-fused positions, we successfully improved fluorescence quantum yield and TPA cross-section simultaneously. Large spectral overlap between energy donor emission and acceptor absorption was achieved and near perfect energy transfer efficiency was acquired for all the studied probes. We revealed that these probes exhibit two well-resolved TPA bands, which are contributed by FRET donors and acceptors, respectively. Especially, both the wavelengths and the cross-sections of the two TPA bands agree well with those of energy donors and acceptors, which is the unique TPA spectral profile of FRET probes and has never been previously reported. Moreover, we proposed an excellent TP-FRET probe BCD3 and its product molecule BCD3-H2S, which exhibit large Stokes (141 nm and 88 nm) and emission shifts (5931 cm-1), as well as greatly increased TP action cross-sections (24-fold and 60-fold) in the near-infrared region with respect to BCD and BCD-H2S. Our detailed study can give an insight into the efficient design of novel TP-FRET fluorescent probes.

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.