Two-photon Fluorescence Imaging Research Articles

Crystallography, Charge Transfer, and Two-Photon Absorption Relations in Molecular Cocrystals for Two-Photon Excited Fluorescence Imaging.

Two-photon excited fluorescence imaging requires high-performance two-photon absorption (TPA) active materials, which are commonly intramolecular charge transfer systems prepared by traditional chemical synthesis. However, this typically needs harsh conditions and new methods are becoming crucial. In this work, based on a collaborative intermolecular charge transfer (inter-CT) strategy, three centimeter-sized organic TPA cocrystals are successfully obtained. All three cocrystals exhibit a mixed stacking arrangement, which can effectively generate inter-CT between the donor and acceptor. The ground and excited state characterizations compare their inter-CT ability: 1,2-BTC > 2D-BTC > 1D-BTC. Transient absorption spectroscopy detects TCNB•-, indicating that the TPA mechanism arises from molecular polarization caused by inter-CT. Meanwhile, 1,2-BTC exhibits the highest excited-state absorption and the longest excited-state lifetime, suggesting a stronger TPA response. First-principles calculations also confirm the presence of inter-CT interactions, and the significant parameter Δµ which can assess the TPA capability indicates that inter-CT enhances the TPA response. Besides, cocrystals also demonstrate excellent water solubility and two-photon excited fluorescence imaging capabilities. This research not only provides an effective method for synthesizing TPA crystal materials and elucidates the connection between inter-CT ability and TPA property but also successfully applies them in the fields of multi-photon fluorescence bioimaging.

Exploring the effect of photobiomodulation and gamma visual stimulation induced by 808 nm and visible LED in Alzheimer's disease mouse model

Although photobiomodulation (PBM) and gamma visual stimulatqion (GVS) have been overwhelmingly explored in the recent time as a possible light stimulation (LS) means of Alzheimer's disease (AD) therapy, their effects have not been assessed at once. In our research, the AD mouse model was stimulated using light with various parameters [continuous wave (PBM) or 40 Hz pulsed visible LED (GVS) or 40 Hz pulsed 808 nm LED (PBM and GVS treatment)]]. The brain slices collected from the LS treated AD model mice were evaluated using (i) fluorescence microscopy to image thioflavine-S labeled amy-loid-β (Aβ) plaques (the main hallmark of AD), or (ii) two-photon excited fluorescence (TPEF) imaging of unlabeled Aβ plaques, showing that the amount of Aβ plaques was reduced after LS treatment. The imaging results correlated well with the results of Morris water maze (MWM) test, which demonstrated that the spatial learning and memory abilities of LS treated mice were noticeably higher than those of untreated mice. The LS effect was also assessed by in vivo nonlinear optical imaging, revealing that the cerebral amyloid angiopathy decreased spe-cifically as a result of 40 Hz pulsed 808 nm irradiation, on the contrary, the angiopathy reversed after visible 40 Hz pulsed light treatment. The obtained results provide useful reference for further optimization of the LS (PBM or GVS) parameters to achieve efficient phototherapy of AD.

Tetraphenylethylene-based AIE nanoprobes for labeling lysosome by two-photon imaging in living cells

Lysosomes are essential cellular organelles, serving vital functions in cellular metabolism and degradation. The design of specifically targeting lysosomes probes with aggregation-induced emission (AIE) characteristics using two-photon excitation techniques is significance and challenging work. Here we designed and synthesized two tetraphenylethylene (TPE)-based AIE fluorescence probes, naming TPE-Ma and TPE-Py, with TPE as the matrix and morpholine (Ma) or pyrrolidone (Py) as the targeting group. These probes exhibit a significant Stokes shift, low cytotoxicity, two-photo fluorescence imaging and lysosome-specific targeting capability ensuring their suitability for fluorescence imaging applications. To enhance the water solubility and cellular accumulation of TPE-Ma and TPE-Py in tumor cells, we employed a biocompatible polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) as a nanocarrier. By encapsulating TPE-Ma and TPE-Py within DSPE-mPEG2000, we successfully developed two AIE fluorescent nanoprobes known as DSPE@ TPE-Ma and DSPE@ TPE-Py. The results demonstrated that fluorescent nanoprobes DSPE@ TPE-Ma and DSPE@ TPE-Py possess excellent cell permeability, biocompatibility, superior photostability and specific targeting towards lysosomes in MCF-7 cells. Our findings highlight the potential of these fluorescent nanoprobes as effective tools for two-photon fluorescence imaging and targeted detection of lysosomes in cancer cells.

Non-invasive assessment of hair regeneration in androgenetic alopecia mice in vivo using two-photon and second harmonic generation imaging.

The identification of crucial targets for hair regrowth in androgenetic alopecia (AGA) involves determining important characteristics and different stages during the process of hair follicle regeneration. Traditional methods for assessing key features and different stages of hair follicle primarily involve taking skin tissue samples and determining them through various staining or other methods. However, non-invasive assessment methods have been long sought. Therefore, in this study, endogenous fluorescence signals from skin keratin and second harmonic signals from skin collagen fibers were utilized as probes, two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging techniques were employed to non-invasively assess hair shafts and collagen fibers in AGA mice in vivo. The TPEF imaging technique revealed that the alternation of new and old hair shafts and the different stages of the growth period in AGA mice were delayed. In addition, SHG imaging found testosterone reduced hair follicle area and miniaturized hair follicles. The non-invasive TPEF and SHG imaging techniques provided important methodologies for determining significant characteristics and different stages of the growth cycle in AGA mice, which will facilitate future non-invasive assessments on human scalps in vivo and reduce the use of animal testing.

Tracing Superoxide Anion in Serotonergic Neurons of Living Mouse Brains with Depression by Small-Molecule Fluorescence Probes.

In brains, the serotonergic neurons are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the physiology of the brain. The dysfunction of serotonergic neurons caused by oxidative stress in the brain is closely related to the occurrence and development of various mental diseases, such as depression. As the biomarker of oxidative stress, the superoxide anion radical (O2•-) can cause oxidative damage to proteins, nucleic acids and lipids, disturbing the function of neurons and brains. A serotonin transporter (SERT) specifically expresses in serotonergic neurons, which is the biomarker of serotonergic neurons. Thus, we created two novel small molecular fluorescent probes (PA-CA and HT-CA) for imaging O2•- in serotonergic neurons of living brains of mice based on specific targeting groups of SERT. Both PA-CA and HT-CA exert excellent SERT-targetable and glorious selectivity for O2•-. Those two probes could monitor the boost of O2•- in living hsert-HEK293 cells that specifically express SERT under oxidative stress. With two-photon fluorescence imaging, we revealed for the first time that O2•- is significantly increased in serotonergic neurons in living brains of mice with depression. More importantly, proteomic analyses suggested that O2•- could oxidize cysteine and histidine in the active site of SERT, which is involved in the development of depression. This work provides new materials for living brain imaging and offers new strategy for unraveling the pathophysiology of depression.

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.

Multimodal imaging of metabolic activities for distinguishing subtypes of breast cancer.

Triple negative breast cancer (TNBC) is a highly aggressive form of cancer. Detecting TNBC early is crucial for improving disease prognosis and optimizing treatment. Unfortunately, conventional imaging techniques fall short in providing a comprehensive differentiation of TNBC subtypes due to their limited sensitivity and inability to capture subcellular details. In this study, we present a multimodal imaging platform that integrates heavy water (D2O)-probed stimulated Raman scattering (DO-SRS), two-photon fluorescence (TPF), and second harmonic generation (SHG) imaging. This platform allows us to directly visualize and quantify the metabolic activities of TNBC subtypes at a subcellular level. By utilizing DO-SRS imaging, we were able to identify distinct levels of de novo lipogenesis, protein synthesis, cytochrome c metabolic heterogeneity, and lipid unsaturation rates in various TNBC subtype tissues. Simultaneously, TPF imaging provided spatial distribution mapping of NAD[P]H and flavin signals in TNBC tissues, revealing a high redox ratio and significant lipid turnover rate in TNBC BL2 (HCC1806) samples. Furthermore, SHG imaging enabled us to observe diverse orientations of collagen fibers in TNBC tissues, with higher anisotropy at the tissue boundary compared to the center. Our multimodal imaging platform offers a highly sensitive and subcellular approach to characterizing not only TNBC, but also other tissue subtypes and cancers.

A piperazine-substituted phthalocyanine with rapid cellular uptake and dual organelle-targeting for in vitro photodynamic therapy

The rational design of photosensitizers with rapid cellular uptake and dual-organelle targeting ability is essential for enhancing the efficacy of photodynamic therapy (PDT). However, achieving this goal is a great challenge. In this paper, a novel axial piperazine substituted (PIP) silicon phthalocyanine (PIP-SiPc) has been synthesized. The PIP substitution significantly improved the cellular uptake of PIP-SiPc in MCF-7 breast cancer cells, as demonstrated by two-photon fluorescence imaging combined with fluorescence correlation spectroscopy. Additionally, PIP-SiPc was able to target both mitochondria and lysosomes simultaneously. Notably, PIP-SiPc exhibited remarkable singlet oxygen generation ability, leading to apoptosis in cancer cells upon irradiation, with an IC50 value of only 0.2 µM. These findings highlight the effectiveness of PIP-SiPc as a multifunctional photosensitizer for PDT.

Dual-Mode Gold Nanocluster-Based Nanoprobe Platform for Two-Photon Fluorescence Imaging and Fluorescence Lifetime Imaging of Intracellular Endogenous miRNA.

Bioimaging is widely used in various fields of modern medicine. Fluorescence imaging has the advantages of high sensitivity, high selectivity, noninvasiveness, in situ imaging, and so on. However, one-photon (OP) fluorescence imaging has problems, such as low tissue penetration depth and low spatiotemporal resolution. These disadvantages can be solved by two-photon (TP) fluorescence imaging. However, TP imaging still uses fluorescence intensity as a signal. The complexity of organisms will inevitably affect the change of fluorescence intensity, cause false-positive signals, and affect the accuracy of the results obtained. Fluorescence lifetime imaging (FLIM) is different from other kinds of fluorescence imaging, which is an intrinsic property of the material and independent of the material concentration and fluorescence intensity. FLIM can effectively avoid the fluctuation of TP imaging based on fluorescence intensity and the interference of autofluorescence. Therefore, based on silica-coated gold nanoclusters (AuNCs@SiO2) combined with nucleic acid probes, the dual-mode nanoprobe platform was constructed for TP and FLIM imaging of intracellular endogenous miRNA-21 for the first time. First, the dual-mode nanoprobe used a dual fluorescence quencher of BHQ2 and graphene oxide (GO), which has a high signal-to-noise ratio and anti-interference. Second, the dual-mode nanoprobe can detect miR-21 with high sensitivity and selectivity in vitro, with a detection limit of 0.91 nM. Finally, the dual-mode nanoprobes performed satisfactory TP fluorescence imaging (330.0 μm penetration depth) and FLIM (τave = 50.0 ns) of endogenous miR-21 in living cells and tissues. The dual-mode platforms have promising applications in miRNA-based early detection and therapy and hold much promise for improving clinical efficacy.

Red fluorescent protein dimer for singlet oxygen (1O2)/ superoxide radical (O2•−) simultaneously mediated photodynamic therapy and two-photon fluorescence imaging

In this work, two lysosome-targeting red fluorescent protein analogues (APFP-lyso and DAPFP-lyso) were synthesized by phenolthiazine and anthracene anchoring strategies for singlet oxygen (1O2)/superoxide radical (O2•−) simultaneously mediated photodynamic therapy (PDT) in A-549 cells and two-photon fluorescence imaging in zebrafish. Compared with APFP-lyso of single-branch structure, fluorescent protein dimer DAPFP-lyso shows larger Stokes shift (Δλ = 5431 cm−1) with near-infrared emission at 664 nm and achieves 1.2- and 10.2-fold enhancement in 1O2 and O2•− production efficiency, respectively. In particular, effective two-photon absorption property of fluorescent protein dimer DAPFP-lyso (δ2PA = 81 GM) imparts the vivid two-photon fluorescence imaging in zebrafish under 800 nm excitation. Theoretical calculation indicated that fluorescent protein dimer DAPFP-lyso shows small singlet-triplet (ΔEST = 0.32 eV) to boost reactive oxygen species production efficiency. Intracellular PDT in A-549 cells, it is worth mentioning that DAPFP-lyso showed good biocompatibility, negligible cell dark toxicity (MTT assay >90%) and high phototoxicity (IC50 = 4.52 μM). The fluorescence imaging in A-549 cells and zebrafish manifests dimer DAPFP-lyso could generate 1O2 and O2•− under irradiation (460 nm, 23 mW cm−2). AO/EB dual staining assays and cell migration experiment in A-549 cells demonstrated that DAPFP-lyso could promote cancer cell apoptosis and migration under irradiation. The fluorescent protein dimer photosensitizer DAPFP-lyso had a precise lysosome-targeting ability (R = 0.95) could recognize lysosome specifically. These experimental results show that the fluorescent protein dimer design is of great significance for the development of new photosensitizers in the field of PDT.

Formalin fixation and paraffin embedding interfere with the preservation of optical metabolic assessments based on endogenous NAD(P)H and FAD two-photon excited fluorescence.

Endogenous NAD(P)H and FAD two-photon excited fluorescence (TPEF) images provide functional metabolic information with high spatial resolution for a wide range of living specimens. Preservation of metabolic function optical metrics upon fixation would facilitate studies which assess the impact of metabolic changes in the context of numerous diseases. However, robust assessments of the impact of formalin fixation, paraffin embedding, and sectioning on the preservation of optical metabolic readouts are lacking. Here, we evaluate intensity and lifetime images at excitation/emission settings optimized for NAD(P)H and FAD TPEF detection from freshly excised murine oral epithelia and corresponding bulk and sectioned fixed tissues. We find that fixation impacts the overall intensity as well as the intensity fluctuations of the images acquired. Accordingly, the depth-dependent variations of the optical redox ratio (defined as FAD/(NAD(P)H + FAD)) across squamous epithelia are not preserved following fixation. This is consistent with significant changes in the 755 nm excited spectra, which reveal broadening upon fixation and additional distortions upon paraffin embedding and sectioning. Analysis of fluorescence lifetime images acquired for excitation/emission settings optimized for NAD(P)H TPEF detection indicate that fixation alters the long lifetime of the observed fluorescence and the long lifetime intensity fraction. These parameters as well as the short TPEF lifetime are significantly modified upon embedding and sectioning. Thus, our studies highlight that the autofluorescence products formed during formalin fixation, paraffin embedding and sectioning overlap highly with NAD(P)H and FAD emission and limit the potential to utilize such tissues to assess metabolic activity.

NIR ditriphenylamine Indole-BODIPY photosensitizer: synthesis, photodynamic therapy in A549 cells and two-photon fluorescence imaging in zebrafish

In this study, the ditriphenylamine Indole-BODIPY photosensitizer T2BDP-lyso was synthesized for near-infrared photodynamic therapy and two-photon fluorescence imaging. The photosensitizer T2BDP-lyso exhibits absorption above 700 nm and emission above 800 nm, respectively. Theoretical calculations show the energy gap from the excited state S1 to the excited state T2 is 0.14 eV, which indicated that the photosensitizer T2BDP-lyso could reach the triplet state by intersystem crossing from the singlet state. Under NIR light, the singlet oxygen yield of photosensitizer T2BDP-lyso was calculated to be 0.64 in CH2Cl2. The photosensitizer T2BDP-lyso can effectively produce reactive oxygen species in A549 cells and zebrafish under 660 nm light for 5 min. The photosensitizer T2BDP-lyso exhibited lower dark toxicity and higher phototoxicity (IC50 = 1.49 μM), as well as lysosomal targeting ability (Pearson coefficient = 0.89). In the AO/EB double staining assay simulating photodynamic therapy at the cellular level, 3 μM of T2BDP-lyso light for 10 min was effective in killing cancer cells. Moreover, the photosensitizer T2BDP-lyso has a large two-photon absorption cross section at 1050 nm, which was calculated to be 138.7 GM in THF by Z-scan method, and two-photon fluorescence imaging was performed in zebrafish. The above results indicate the potential application of the photosensitizer T2BDP-lyso in near-infrared photodynamic therapy and two-photon fluorescence imaging.

Investigating biochemical and structural changes of glycated collagen using multimodal multiphoton imaging, Raman spectroscopy, and atomic force microscopy

Advanced glycation end products (AGEs) form extracellular crosslinking with collagenous proteins, which contributes to the development of diabetic complications. In this study, AGEs-related pentosidine (PENT) crosslinks-induced structural and biochemical changes are studied using multimodal multiphoton imaging, Raman spectroscopy and atomic force microscopy (AFM). Decellularized equine pericardium (EP) was glycated with four ribose concentrations ranging between 5 and 200 mM and monitored for up to 30 days. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopic imaging probed elastin and collagen fibers, respectively. The glycated EP showed a decrease in the SHG intensities associated with loss of non-centrosymmetry of collagen and an increase of TPEF intensities associated with PENT crosslinks upon glycation. TPEF signals from elastin fibers were unaffected. A three-dimensional reconstruction with SHG + TPEF z-stack images visualized the distribution of collagen and elastin within the EP volume matrix. In addition, Raman spectroscopy (RS) detected changes in collagen-related bands and discriminated glycated from untreated EP. Furthermore, AFM scans showed that the roughness increases and the D-unit structure of fibers remained unchanged during glycation. The PENT crosslinked-induced changes are discussed in the context of previous studies of glutaraldehyde- and genipin-induced crosslinking and collagenase-induced digestion of collagen. We conclude that TPEF, SHG, RS, and AFM are effective, label-free, and non-destructive methods to investigate glycated tissues, differentiate crosslinking processes, and characterize general collagen-associated and disease-related changes, in particular by their RS fingerprints.

Fluorescent protein chromophore-based photosensitizer modified with anthracenyl for lysosome-targeted photodynamic therapy and two-photon fluorescence imaging

In this work, two cis-anthracenyl fluorescent protein chromophore analogs, AnFP-1 and AnFP-2 were synthesized by [2 + 3] cycloaddition for photodynamic therapy and two-photon fluorescence imaging. We combine the anthracenyl group and fluorescent core imidazolinone to form the dye AnFP-1, and then the morpholine indole structure was introduced via Knoevenagel reaction to obtain the dye AnFP-2. The heavy-atom-free photosensitizing dyes AnFP-1 and AnFP-2 have satisfied yields of singlet oxygen, measured to be 30% and 21% in methanol solution, respectively. The tetrazolium blue (MTT) experiment showed that the survival rate of A549 cells was over 92% without light exposure and under 460 nm light exposure for 10 min, the half-inhibitory concentrations (IC50) of the photosensitizing dyes AnFP-1 and AnFP-2 were 3.7 μmol•L−1 and 4.1 μmol•L−1, respectively. Acridine orange (AO)/Ethidium bromide (EB) double staining experiments showed that the two photosensitizing dyes could generate singlet oxygen and induce apoptosis in cancerous cells under irradiation. In addition, the photosensitizing dye AnFP-2 has high specificity to lysosomes which improve the photodynamic effect. Its Pearson co-localization correlation coefficient is 93%. In biological imaging, AnFP-1 and AnFP-2 showed satisfactory two-photon fluorescence imaging in zebrafish with 800 nm femtosecond laser excitation. According to these results, AnFP-1 and AnFP-2 can be used in photodynamic therapy and two-photon fluorescence imaging, which lays the foundation for developing new photosensitizing dyes.

Hollow Core Double-Clad Fiber Coupler for Nonlinear Micro-Endoscopy

We report the design, fabrication and characterization of the first hollow core double-clad fiber coupler for nonlinear micro-endoscopy. It is made of a pure silica negative curvature hollow core double-clad fiber and a highly multimode fiber fused along each other. The fabrication of such a coupler was made possible thanks to the use of a soft (borosilicate) glass multimode fiber which can be fused along the negative curvature fiber without altering its geometry, thanks to its much lower fusion temperature. A 60% power transfer from the double-clad fiber to the multimode one was measured at 500 nm. Demonstration of two-photon fluorescence and second harmonic generation imaging was performed on unlabeled biological tissues with an endoscopic head mounted on the hollow core double-clad fiber coupler to demonstrate its applicability. This device greatly simplifies the integration of nonlinear micro-endoscopes based on hollow core double clad fibers.

An acridone-derived fluorescent off-on probe for detection and in vivo imaging of nitroreductase

Understanding the functions of enzymes in various physiological processes is important, but the design of signaling probes for fast analysis of enzymatic activity is particularly challenging. Herein, a fluorescence-enhanced probe, 10-methyl-2-nitro-acridone (MNA), was synthesized and applied to analyze nitroreductase (NTR) activity in vitro and in vivo. The detection mechanism is based on the nitro group in MNA reacting toward NTR with high reactivity and generating 10-methyl-2-amino- acridone (MAA) accompanied by an obvious fluorescence signal enhancement at 525 nm emission. The probe shows low cytotoxicity, fast response, and high selectivity and sensitivity with a limit of detection as low as 150 ng·mL−1. The probe was also employed for two-photon fluorescence imaging of NTR in zebrafish in vivo revealing the distribution of NTR. Versus existing NTR probes, the proposed probe shows favorable analytical performance including near-infrared light excitation with no other byproducts produced after the reaction. The superior properties of this signaling probe allow it to become a fluorescence imaging candidate in other biosystems.

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.

Accurate Detection of Hcy in Human Serum and Two-Photon Visualization of Atherosclerosis Using a Highly Specific Fluorescent Probe.

As an important metabolic intermediate of sulfur-containing amino acids in human body, homocysteine (Hcy) is regarded as an independent risk factor for atherosclerotic cardiovascular disease. Therefore, real-time monitoring of the fluctuation of Hcy level is of great importance for the early diagnosis as well as the treatment of atherosclerosis. Herein, a new two-photon (TP) fluorescent probe (RH-2) was developed via a hydrogen bond-assisted strategy, which had a high specificity for detecting Hcy over cysteine (Cys) and glutathione (GSH) in solution, cells, and tissue. Probe RH-2 was applied to the quantitative determination of Hcy in human serum successfully. Moreover, the two-photon fluorescence (TPF) imaging of abnormal expression of Hcy in aortic vessels and liver of atherosclerotic model mice were fulfilled by RH-2. Therefore, probe RH-2 can be served as a potential tool to understand the function of Hcy in atherosclerosis, supplying a clinical promise for the early diagnosis of atherosclerosis (AS).

Photostable organic two-photon dyes with ultrahigh brightness for long-term fluorescence imaging.

Developing photostable two-photon dyes with high brightness and negligible photocytotxicity is of key importance in fluorescence imaging but remains a huge challenge. Here, a two-photon dye with ultrahigh brightness and photostability is demonstrated for high-performance long-term two-photon fluorescence imaging. By terminated donor engineering, the designed DBD shows higher two-photon absorption cross-section (δ, 418 GM vs 329 GM) and photoluminescence quantum yield (ΦPL, 62.74% vs 54.63%) than counterpart DBA. As a consequence, two-photon fluorescence brightness (δ×ΦPL) of DBD exhibits a 10-folded enhancement (262 GM vs 19 GM) in comparison with typical Coumarin 307 dye. More importantly, DBD present ultrahigh photostability and negligible photobleaching under 10 min femtosecond laser irradiation, which stands marked contrast to Coumarin 307. Furthermore, femtosecond transient absorption spectroscopy ascribes this ultrahigh photostability and negligible photobleaching to the inefficient intersystem crossing. With these merits, DBD perform long-term two-photon fluorescence imaging in vitro.

Carbonized Polymer Dot Probe for Two-Photon Fluorescence Imaging of Lipid Droplets in Living Cells and Tissues.

As a dynamic and multifunctional organelle, lipid droplets (LDs) are essential in maintaining lipid balance and transducing biological signals. LD accumulation and catabolism are closely associated with energy metabolism and cell signaling. In order to easily trace LDs in living cells, a novel carbonized polymer dot (CPD)-based fluorescent nanoprobe is reported to serve the needs of LD-targeting imaging. This probe exhibits the advantages of excellent biocompatibility, simple preparation, good lipophilicity, and high compatibility with commercial dyes. Transient absorption spectroscopy was employed to discuss the luminescence mechanism of CPDs, and the results indicate that the excellent fluorescence property and the environment-responsive feature of our CPDs are derived from the intramolecular charge transfer (ICT) characteristics and the D-π-A structure that possibly formed in CPD. This nanoprobe is available for one-photon fluorescence (OPF) and two-photon fluorescence (TPF) imaging and is also practicable for staining LDs in living/fixed cells and lipids in tissue sections. The staining process is completed within several seconds, with no washing step. The intracellular LDs involving the intranuclear LDs (nLDs) can be selectively lit up. This probe is feasible for visualizing dynamic interactions among LDs, which suggests its great potential in revealing the secret of LD metabolism. The in situ TPF spectra were analyzed to determine surrounding microenvironment according to the polarity-responsive feature of our CPDs. This work expands the applications of CPDs in biological imaging, helps design new LD-selective fluorescent probes, and has implications for studying LD-related metabolism and diseases.