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Cell Imaging Using Two-Photon Excited CdS Fluorescent Quantum Dots Working within the Biological Window
摘要： In recent years, two-photon excited semiconductor quantum dots (QDs) have been the subject of intense investigation due to their long excitation wavelength which helps to achieve deeper penetration and higher image resolution in optical bioimaging. In this paper, water-soluble CdS QDs were synthesized using a hydrothermal method and applied to human liver hepatocellular carcinoma (HepG2) cells. The first-principles calculation suggested that the S-rich defected structure contributes to a narrower band gap compared to the pristine structure. The resulting fluorescence wavelength was significantly red shifted, which was attributed to the deep defect states emission. The large Stokes shifts (> 200 nm) of the QDs can eliminate the possible cross-talk between the excitation light and the emission light. Two-photon induced red fluorescence emission can avoid overlapping with the autofluorescence emission of biological samples. The uptake and cell viability measurements of the HepG2 cells showed a good biocompatibility and a low toxicity of CdS QDs. Two-photon excited scanning microscopy images revealed that the HepG2 cells incubated with CdS QDs emitted bright red upconversion fluorescence and the fluorescence brightness was 38.2 times of that of the control group. These results support CdS QDs as a good candidate for application in cellular imaging.
关键词： two-photon absorption,CdS quantum dots,deep defect states,HePG2 cells,biological imaging
更新于2025-11-21 11:24:58
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Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques
摘要： PURPOSE. To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. METHODS. Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 lm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. RESULTS. Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm. CONCLUSIONS. Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.
关键词： second-harmonic generation,aqueous outflow,nonlinear microscopy,glaucoma,two-photon fluorescence microscopy
更新于2025-11-21 11:08:12
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<i>In vivo</i> imaging reveals reduced activity of neuronal circuits in a mouse tauopathy model
摘要： Pathological alterations of tau protein play a significant role in the emergence and progression of neurodegenerative disorders. Tauopathies are characterized by detachment of the tau protein from neuronal microtubules, and its subsequent aberrant hyper-phosphorylation, aggregation and cellular distribution. The exact nature of tau protein species causing neuronal malfunction and degeneration is still unknown. In the present study, we used mice transgenic for human tau with the frontotemporal dementia with parkinsonism-associated P301S mutation. These mice are prone to develop fibrillar tau inclusions, especially in the spinal cord and brainstem. At the same time, cortical neurons are not as strongly affected by fibrillar tau forms, but rather by soluble tau forms. We took advantage of the possibility to induce formation of neurofibrillary tangles in a subset of these cortical neurons by local injection of preformed synthetic tau fibrils. By using chronic in vivo two-photon calcium imaging in awake mice, we were able for the first time to follow the activity of individual tangle-bearing neurons and compare it to the activity of tangle-free neurons over the disease course. Our results revealed strong reduction of calcium transient frequency in layer 2/3 cortical neurons of P301S mice, independent of neurofibrillary tangle presence. These results clearly point to the impairing role of soluble, mutated tau protein species present in the majority of the neurons investigated in this study.
关键词： neurofibrillary tangles,two-photon imaging,tau,seeding,P301S mice
更新于2025-11-21 11:08:12
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Screening of two-photon activated photodynamic therapy sensitizers using a 3D osteosarcoma model
摘要： Photodynamic therapy (PDT) involves a photosensitizing agent activated with light to induce cell death. Two-photon excited PDT (TPE-PDT) offers numerous benefits compared to traditional one-photon induced PDT, including an increased penetration depth and precision. However, the in vitro profiling and comparison of two-photon photosensitizers (PS) are still troublesome. Herein, we report the development of an in vitro screening platform of TPE-PS using a 3D osteosarcoma cell culture. The platform was tested using three different two-photon (2P) active compounds – a 2P sensitizer P2CK, a fluorescent dye Eosin Y, and a porphyrin derivative (TPP). Their 2P absorption cross-sections (σ2PA) were characterised using a fully automated z-scan setup. TPP exhibited a remarkably high σ2PA at 720 nm (8865 GM) and P2CK presented a high absorption at 850 nm (405 GM), while Eosin Y had the lowest 2P absorption at the studied wavelengths (<100 GM). The cellular uptake of PS visualized using confocal laser scanning microscopy showed that both TPP and P2CK were internalized by the cells, while Eosin Y stayed mainly in the surrounding media. The efficiency of the former two TPE-PS was quantified using the PrestoBlue metabolic assay, showing a significant reduction in cell viability after two-photon irradiation. The possibility of damage localization was demonstrated using a co-culture of adipose derived stem cells together with osteosarcoma spheroids showing no signs of damage to the surrounding healthy cells after TPE-PDT.
关键词： two-photon excited photodynamic therapy,PrestoBlue assay,photosensitizers,cellular uptake,localized damage,z-scan,3D osteosarcoma model
更新于2025-11-21 11:08:12
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Luminescent Mesoporous Silica Nanohybrid Based on Drug Derivative Terbium Complex
摘要： Mesoporous silica nanoparticles prepared by organic template-driven synthesis have been successfully explored as carriers of the drug-derivate green luminescent complex of terbium (III) with the nonsteroidal anti-inflammatory drug ketoprofen. The terbium (III) complex was synthesized by reacting ketoprofen sodium salt with terbium (III) chloride, which was further adsorbed onto the surface of mesoporous nanoparticles with a mean particle size of 47 ± 4 nm and pore size of 11 nm. The incorporation of the complex into mesoporous silica nanoparticles was tracked by the decrease in the surface area and pore size of the nanoparticles, and successfully demonstrated by substantial changes in the adsorption isotherms and thermal and vibrational spectroscopy results. The cytotoxicity assay and confocal microscopy have shown that the novel luminescent nanohybrid presents high cell viability and the characteristic terbium (III) emission can be assessed through two-photon excitation, which paves the way for bioimaging applications in nanomedicine.
关键词： ketoprofen,mesoporous silica nanoparticles,terbium,luminescent nanohybrid,two-photon
更新于2025-11-21 11:08:12
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Two-photon excited organic nanoparticles for chemo-photodynamic therapy
摘要： Drug molecules assembling nanomedicines possess several advantages, including precise molecular structure, various combinations of theraputic agents and high content of drugs. In this work, paclitaxel dimer and two-photo photosensitizer were devised and synthesized, which could coassemble into nanoparticles (Co-NPs) in aqueous medium through nanopreicipitation method. As-synthesized Co-NPs possess the uniform size of about 80 nm and great stability in physiological condition, and could produce the singlet oxygen upon near-infrared light irradiation. The Co-NPs indicate enhanced cellular uptake and endosomal escape upon irradiation, which result in the synergistic enhancement of cytotoxicity towards cancer cells and growth inhibition of human cervical cancer tumors. We believed this combination therapy based on organic nanoparticles represent a new and important development in the cancer therapy.
关键词： two-photon,photodynamic therapy,paclitaxel,chemotherapy
更新于2025-11-21 11:08:12
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<p>Biodistribution and sensitive tracking of immune cells with plasmonic gold nanostars</p>
摘要： Aim: To quantitatively and sensitively investigate the biodistribution of immune cells after systemic administration. Methods: Immune cells were loaded with plasmonic gold nanostars (GNS) tracking probes. Inductively coupled plasma mass spectrometry (ICP-MS) was used for quantitative gold mass measurement and two-photon photoluminescence (TPL) was used for high-resolution sensitive optical imaging. Results: GNS nanoparticles were loaded successfully into immune cells without negative effect on cellular vitality. Liver and spleen were identified to be the major organs for macrophage cells uptake after systematic administration. A small amount of macrophage cells were detected in the tumor site in our murine lymphoma animal model. Conclusion: GNS has great potential as a biocompatible marker for quantitative tracking and high-resolution imaging of immune cells at the cellular level.
关键词： biodistribution,immune cells,GNS,ICP-MS,two-photon microscopy
更新于2025-11-19 16:56:42
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dz2 Orbitals Mediated Bound Magnetic Polarons in Ferromagnetic Ce Doped BaTiO3 Nanoparticles and its Enriched Two Photon Absorption Cross Section
摘要： Enriched ferromagnetism and two photon absorption (TPA) cross section of perovskite BaTiO3 nanoparticles are indispensible for magnetic and optical data storage applications. In this work, the hydrothermally synthesized Ce doped BaTiO3 nanoparticles exhibit the maximum room temperature ferromagnetism (4.26×10-3 emu/g) at 4 mol% due to the increase of oxygen vacancies as evidenced by X-ray photoelectron, electron spin resonance spectroscopies and density functional theory (DFT) calculations. Hence, the oxygen vacancy constituted bound magnetic polaron (BMP) model has been invoked to explain the enhancement of ferromagnetism. BMP theoretical model indicates the increase of BMP magnetization (M0, 3.0 to 4.8×10-3 emu/g) and true spontaneous moment per BMP (meff, 4 to 9.88×10-4 emu) on Ce doping. DFT calculations show that BMPs mediate via Ti d orbitals leading to the ferromagnetism. Besides, it is understood that the magnetic moment induced by Ce at Ba site is higher than Ce at Ti site in the presence of oxygen vacancies. Open aperture Z-scan technique displays the highest TPA coefficient β (7.08×10-10 m/W) and TPA cross section σTPA (455×104 GM) at 4 mol% of Ce as a result of robust TPA induced excited state absorption. A large σTPA is attributed to the longer excited state lifetime τ (7.63 ns) of charge carriers created by oxygen vacancies and Ce ions which encounter several electronic transitions in the excited sub-states.
关键词： Ce doping,oxygen vacancies,bound magnetic polarons,two photon absorption,DFT calculations,Z-scan technique,BaTiO3 nanoparticles,ferromagnetism
更新于2025-11-19 16:56:35
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Photostable Ratiometric Two-photon Fluorescent Probe for Visualizing Hydrogen Polysulfide in Mitochondria and Its application
摘要： Hydrogen polysulfide (H2Sn) has currently attracted much research interest because it not only plays important physiological function in many biological and health-related events, but also considered as a newfound potent signal transducer. Small-molecule based ratiometric fluorescent probes have advantages in sensitivity and bio-detections but such approaches that intentionally developed for H2Sn detection expected to be mitochondria-accessible are still lacking. In this work, due to that triphenylphosphine group introduced into the molecular scaffold of naphthalimide derivative, Mito-NRT-HP was successfully applied to visualize intracellular H2Sn in mitochondria with excellent aqueous solubility, super photobleaching resistance, favorable cellular membrane permeability and good biocompatibility. This one- and two-photon fluorescent probe with high selectivity and sensitivity (LOD = 0.01 μM) evinced 70-fold enhancement of fluorescence ratio (I546 nm/I478 nm) in the presence of H2Sn over other reactive sulfur species (RSS). The experimental results also give Mito-NRT-HP the potential for mapping the H2Sn distribution in mitochondria and evaluating the H2Sn roles in more biological processes and demonstrated the practical application possibility of Mito-NRT-HP in early diagnosis of LPS-induced acute organ injury.
关键词： mitochondria,fluorescent probe,hydrogen polysulfide,ratiometric,two-photon
更新于2025-11-14 15:29:11
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Photo-physical properties of substituted 2,3-distyryl indoles: Spectroscopic, computational and biological insights
摘要： The structural dependence of the photo-physical properties of substituted 2,3-distyryl (23DSI) indoles were studied using several spectroscopic techniques including steady-state UV-VIS spectroscopy, steady-state fluorescence spectroscopy, steady-state excitation spectroscopy, time correlated single photon counting (TCSPC) spectroscopy, and time-resolved fluorescence upconversion spectroscopy (TRFLS). Each of 23DSI derivatives investigated showed distinct fluorescence emission and UV-VIS spectra, indicating strong structural dependence of the emission and the excitation. The UV-VIS spectra of the 23DSI derivatives showed three main identical absorption bands with minor deviations in the absorbance caused by substituent groups on the distyryl rings. The time-resolved fluorescence up-conversion studies indicated that the fluorescence undergoes a mono-exponential decay whereas the calculated fluorescence lifetime showed relatively short fluorescence lifetimes of approximately 1 ns. All of the 23DSI derivatives showed two-photon absorption upon direct excitation of 1.6 W laser pulses at 800 nm. These studies suggest that the substituents, attached to distyryl core, are capable of boosting or hindering fluorescence intensities by distorting the π-conjugation of the 23DSI molecule. Our studies showed that 23DSI (p-F) has the highest fluorescence emission quantum yield. Theoretical calculations for the ground state of 23DSI derivatives confirmed differences in electron densities in 23DSI derivatives in the presence of different substituent attachments. The excellent fluorescence emission, high fluorescence quantum yield and two-photon absorption properties of these 23DSI molecules make them attractive candidates for potential applications in the fields of biological imaging, biomedicine, fluorescent probes, and photodynamic inactivation (PDI). B. subtilis samples, treated with micro molar solutions of 23DSI (p-OCH3) and 23DSI (p-CH3), showed very effective photodynamic inactivation (PDI) upon irradiation with white light.
关键词： Two photon absorption,Time-resolved fluorescence upconversion laser spectroscopy,Photodynamic inactivation,Gaussian calculations,Photo-physical properties,Light-activation
更新于2025-11-14 15:26:12