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The World Health Organization (WHO) cancer agency predicts that more than 35 million cases of cancer will be experienced in 2050, a 77% increase over the 2022 estimate. Currently, the main cancers diagnosed are breast, lung, and colorectal. There is no standardized tool for cancer diagnoses; initially, clinical procedures are guided by the patient symptoms and usually involve biochemical blood tests, imaging, and biopsy. Label-free non-linear optical approaches are promising tools for tumor imaging, due to their inherent non-invasive biosafe contrast mechanisms and the ability to monitor collagen-related disorders, and biochemical and metabolic changes during cancer progression. In this review, the main non-linear microscopy techniques are discussed, according to three main contrast mechanisms: biochemical, metabolic, and structural imaging. [ABSTRACT FROM AUTHOR]

The World Health Organization (WHO) cancer agency predicts that more than 35 million cases of cancer will be experienced in 2050, a 77% increase over the 2022 estimate. Currently, the main cancers diagnosed are breast, lung, and colorectal. There is no standardized tool for cancer diagnoses; initially, clinical procedures are guided by the patient symptoms and usually involve biochemical blood tests, imaging, and biopsy. Label-free non-linear optical approaches are promising tools for tumor imaging, due to their inherent non-invasive biosafe contrast mechanisms and the ability to monitor collagen-related disorders, and biochemical and metabolic changes during cancer progression. In this review, the main non-linear microscopy techniques are discussed, according to three main contrast mechanisms: biochemical, metabolic, and structural imaging.

Two‐photon excitation (2PE) represents substantial advantages in biophotonics over traditional one‐photon excitation (1PE), offering enhanced spatial resolution and deeper tissue penetration capabilities. Since its introduction in the 1990s, two‐photon excited microscopy has spurred the rapid emergence of novel 2PE‐based applications, including photodynamic therapy (PDT) and the targeted release of biologically active agents. This review first elucidates the fundamental principles of 2PE before comprehensively examining the evolution of photosensitizers (PSs) for two‐photon PDT (2P‐PDT), covering both nanomaterials and small molecules, categorized based on their respective characteristics. Additionally, it explores advancements in PS development for integrating 2P‐PDT with other therapeutic modalities (e.g., photothermal therapy, chemotherapy, immunotherapy, gene therapy, etc.). Subsequent sections provide an overview of photoactivatable compounds with two‐photon absorption properties for the controlled release of various therapeutic agents (e.g., drugs, genes, and gasotransmitters), emphasizing their potential in anticancer applications. Last, a deep discussion deciphers the prospects and challenges of 2P‐mediated therapy in cancer treatment, particularly concerning their clinical translation. [ABSTRACT FROM AUTHOR]

The low signal-to-noise ratio (S/N) and single functionality of fluorescence imaging agents have limited their practical applications. Bright two-photon excitation (2PE) imaging probes are highly desirable in vivo with larger imaging depth, minor autofluorescence background, and less photodamage. Herein, we developed responsive aggregated gold nanoparticles (Au NPs) as high contrast 2PE imaging agents, capable of emitting red fluorescence upon excitation of near-infrared (NIR) laser and possessing a high signal-to-noise ratio (S/N of 2,475). By forming aggregates in situ inside tumor tissue, doxorubicin hydrochloride (DOX)-loaded mix-charged gold nanoparticles (DOX-MC-Au NPs) were utilized to act as selective fluorescence imaging probes and precise therapy agents. These high-contrast theranostic agents offer a promising potential for precise cancer imaging and therapy, which might open a new venue to multifunctional and noninvasive theranostics. [ABSTRACT FROM AUTHOR]

The surface of Eu0.3La0.7F3 submicron particles is functionalized with a blue‐emitting Ir(III) complex (1) specifically designed to coordinate lanthanide(III) ions efficiently through a carboxylic unit. In this Eu0.3La0.7F3@1 composite, the Ir(III) complexes are randomly coordinated to either superficial La(III) ions or Eu(III) ions (Ir‐Lasurface and Ir‐Eusurface pairs, respectively) and its color emission, a balance between blue (Ir‐based) and red (Eu‐based) light, can be tuned as a function of the excitation wavelength. Irradiation at the maximum of the excitation band in 1 promotes blue emission from Ir‐Lasurface pairs and red emission from Eusurface ions sensitized by Ir → Eusurface energy transfer (EnT). At λexc = 396 nm (maximum of the Eu(III) 5L6 ← 7F0 absorption band) the red emission from inner Eu(III) ions becomes dominant. Excitation of 1 can also be achieved by two photon‐absorption (TPA) since this complex has a moderate cross‐section of σ2 = 9.4 ± 1.0 GM at 780 nm (σ2 = 5.8 ± 0.6 GM at 800 nm). Phosphorescence lifetime imaging (PLIM) allows the emission of individual particles to be visualized and the Ir‐, Eusurface‐, and Eucore‐based emissions can be distinguished due to the significant differences in their respective emission lifetimes. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a minimally invasive treatment for several diseases. It combines light energy with a photosensitizer (PS) to destroy the targeted cells or tissues. A PS itself is a non-toxic substance, but it becomes toxic to the target cells through the activation of light at a specific wavelength. There are some limitations of PDT, although it has been used in clinical studies for a long time. Two-photon excitation (TPE) and upconversion (UC) for PDT have been recently developed. A TPE nanoparticle-based PS combines the advantages of TPE and nanotechnology that has emerged as an attractive therapeutic agent for near-infrared red (NIR) light-excited PDT, whilst UC is also used for the NIR light-triggered drug release, activation of 'caged' imaging, or therapeutic molecules during PDT process for the diagnosis, imaging, and treatment of cancers. Methods: Nine electronic databases were searched, including WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and China National Knowledge Infrastructure (CNKI), without any language constraints. TPE and UCNP were evaluated to determine if they had different effects from PDT on cancers. All eligible studies were analyzed and summarized in this review. Results: TPE-PDT and UCNP-PDT have a high cell or tissue penetration ability through the excitation of NIR light to activate PS molecules. This is much better than the conventional PDT induced by visible or ultraviolet (UV) light. These studies showed a greater PDT efficacy, which was determined by enhanced generation of reactive oxygen species (ROS) and reduced cell viability, as well as inhibited abnormal cell growth for the treatment of cancers. Conclusions: Conventional PDT involves Type I and Type II reactions for the generation of ROS in the treatment of cancer cells, but there are some limitations. Recently, TPE-PDT and UCNP-PDT have been developed to overcome these problems with the help of nanotechnology in in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Open shell species are alluring significant attention owing to their unique physiochemical properties in redox chemistry for activating remarkably stable bonds. Solvated electrons are one of them that have been extensively investigated due to their high reduction potential (Ered=−2.9 V vs SHE in CH3CN), diverse substrate activation, and promising applications. If the activating species have a larger redox potential with a longer lifetime, then the broader range of substrates will be activated. Hence, the solvated electron qualifies as a super‐reductant with these qualities. However, due to safety issues, generating solvated electrons by dissolving alkali metals in an ammoniated solvent limits its use towards complicated organic transformations. Instead photochemically generated solvated electron overcome this limitation and is identified as a user‐friendly, sustainable, and much safer alternative approach for producing solvated electrons. In this minireview, we have comprehensively highlighted the recent key methods to generate the solvated electron photochemically, characterization techniques, and its application in organic transformations with selected examples. The minireview provides new opportunities for chemists to understand the conceptual, physical, and mechanistic chemistry principle of this super reductant for exploiting a new photochemical route for the transformations that are difficult to achieve by other means. [ABSTRACT FROM AUTHOR]

Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using onephoton excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49;300 M-1 cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusions: T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein (BMP) signaling plays a vital role in differentiation, organogenesis, and various cell processes. As a member of TGF‐β superfamily, the BMP initiation usually accompanies crosstalk with other signaling pathways and simultaneously activates some of them. It is quite challenging to solely initiate an individual pathway. In this study, an opsin‐free optical method to specifically activate BMP receptors (BMPR) and subsequent pSmad1/5/8 cascades by a single‐time scan of a tightly‐focused femtosecond laser in the near infrared range is reported. Via transient two‐photon excitation to intrinsic local flavins near the cell membrane, the photoactivation drives conformational changes of preformed BMPR complexes to enable their bonding and phosphorylation of the GS domain in BMPR‐I by BMPR‐II. The pSmad1/5/8 signaling is initiated by this method, while p38 and pSmad2 are rarely perturbed. Based on a microscopic system, primary adipose‐derived stem cells in an area of 420 × 420 µm2 are photoactivated by a single‐time laser scanning for 1.5 s and exhibit pSmad1/5/8 upregulation and osteoblastic differentiation after 21 days. Hence, an opsin‐free, specific, and noninvasive optical method to initiate BMP signaling, easily accomplished by a two‐photon microscope system is reported. [ABSTRACT FROM AUTHOR]

Time-resolved photoluminescence measurements were conducted on ZnO single crystals using two-photon (2P) excitation at low temperatures ranging from 25 to 260 K. The decay constants of various emission peaks are determined and compared between one-photon and two-photon excitations using a recently modified localized-state ensemble model. The analysis is focused on the redshift of the phonon-assisted free exciton transition energy with increasing temperature. A significantly longer radiative recombination lifetime of approximately 840 ps is observed under 2P excitation. The extended lifetime of excitons during 2P excitation is achieved through the interplay of multiple mechanisms. Photon-recycling, enabled by high absorption coefficients and total internal reflection, facilitates re-absorption and new carrier generation. Phonon scattering lowers photon energy, allowing their escape from the crystal, while exciton–polariton interactions delay photon travel, collectively contributing to the extension of exciton lifetimes. The re-absorption effect and elongated carrier lifetime observed from our work could be beneficial for applications of ZnO materials in 2P imaging, particularly of biological samples. [ABSTRACT FROM AUTHOR]

Rydberg atoms possess highly excited valence electrons that are far away from atomic cations. Compared with ground states, Rydberg states are excited states with a high principal quantum number n that exhibit large electric dipole moments and have a variety of applications in quantum information processing. In this communication, we report the measurement of the 6 Li Rydberg excitation spectrum by ladder-type electromagnetically induced transparency (EIT) in a vapor cell. The 2 p → n s / n d EIT spectra were recorded by sweeping the frequency of an ultraviolet Rydberg pumping laser while keeping the probing laser resonant to the 2 s → 2 p transition. All lasers were locked on an ultrastable optical Fabry-Pérot cavity and measured by an optical frequency comb. Our results provide valuable information to precisely determine quantum defects and enable novel experiments with Rydberg-dressed ultracold Fermi gases. [ABSTRACT FROM AUTHOR]

The multiple metastable excited states provided by excited‐state intramolecular proton transfer (ESIPT) molecules are beneficial to bring temperature‐dependent and color‐tunable long persistent luminescence (LPL). Meanwhile, ESIPT molecules are intrinsically suitable to be modulated as D‐π‐A structure to obtain both one/two‐photon excitation and LPL emission simultaneously. Herein, we report the rational design of a dynamic CdII coordination polymer (LIFM‐106) from ESIPT ligand to achieve the above goals. By comparing LIFM‐106 with the counterparts, we established a temperature‐regulated competitive relationship between singlet excimer and triplet LPL emission. The optimization of ligand aggregation mode effectively boost the competitiveness of the latter. In result, LIFM‐106 shows outstanding one/two‐photon excited LPL performance with wide temperature range (100–380 K) and tunable color (green to red). The multichannel radiation process was further elucidated by transient absorption and theoretical calculations, benefiting for the application in anti‐counterfeiting systems. [ABSTRACT FROM AUTHOR]

Non-radiative traps and structures are present on the graphene quantum dots doped with nitrogen and functionalized with amino groups (amino-N-GQDs) and multiple crystalline layers because of cross-link-enhanced emission. Secondary and tertiary amines, which are potential fluorophores, have also been observed on the polyethylenimine (PEI) coating of amino-N-GQDs. Cross-linked PEI coating reduced rotation and vibration, thereby enhancing the photoluminescence quantum yield (PL QY). Introduced nitrogen atoms from N dopants, amino-functionalized groups and PEI, as well as sulfur from polystyrene sulfonate (PSS) enhanced the cooperative effect on the properties of heteroatom-doped materials among electrons captured by new surface states. This enhanced radiative recombination and subsequently enhanced PL QY, therefore, surface conjugation improved the amino-N-GQD surfaces by increasing the quantum confinement of their emissive energy, evidenced by the increased PL QY of amino-N-GQD-PSS-PEI (or amino-N-GQD-polymer composites). In some situations, the maximum available power required for delivery to the two-photon imaging plane without damaging tissues limits imaging depth but the additional brightness provided by amino-N-GQD-polymer composites in this study extended the maximum imaging depth to 240 μm. Amino-N-GQD-polymer composites had favorable two-photon properties under two-photon excitation (self-developed femtosecond Ti–sapphire laser optical system; power: 23.93 nJ pixel−1, 160 scans, approximately 1.09 s of total exposure time; excitation wavelength: 980 nm, near-infrared-II region), indicating that cells treated with amino-N-GQD-polymer composites and the anti-epidermal growth factor receptor antibody can achieve two-photon luminescence with 1/81 of the power required for similar-intensity two-photon autofluorescence (1938.33 nJ pixel−1 with 800 scans, total exposure time of ∼5.44 s). The materials can serve as contrast agents for the non-invasive detection of biological specimens and interior tissues using a two-photon excitation wavelength in the near-infrared region.

Photoactivatable fluorophores (PAFs) are powerful tools for biological imaging applications because they provide spatiotemporal control of fluorescence distribution. Many of the existing PAFs can only be activated by UV irradiation. In our study, we present a blue light (1P) and NIR light (2P) activatable rhodamine fluorophore. Next to the description of the synthesis and the investigation of the photoreaction, we demonstrate the use of our PAF in the context of laser scanning microscopy. By immobilization of our PAF in a hydrogel, we were able to write and read spatially resolved illumination patterns with excellent contrast after both one‐photon and two‐photon excitation. [ABSTRACT FROM AUTHOR]

Two-photon excitation in light-sheet microscopy advances applications to live imaging of multicellular organisms. In a previous study, we developed a two-photon Bessel beam light-sheet microscope with a nearly 1-mm field of view and less than 4-μm axial resolution, using a low magnification (10×), middle numerical aperture (NA 0.5) detection objective. In this study, we aimed to construct a light-sheet microscope with higher resolution imaging while maintaining the large field of view, using low magnification (16×) with a high NA 0.8 objective. To address potential illumination and detection mismatch, we investigated the use of a depth of focus (DOF) extension method. Specifically, we used a stair-step device composed of five-layer annular zones that extended DOF two-fold, enough to cover the light-sheet thickness. Resolution measurements using fluorescent beads showed that the reduction in resolutions was small. We then applied this system to in vivo imaging of medaka fish and found that image quality degradation at the distal site of the beam injection could be compensated. This demonstrates that the extended DOF system combined with wide-field two-photon light-sheet microscopy offers a simple and easy setup for live imaging application of large multicellular organism specimens with sub-cellular resolution. [ABSTRACT FROM AUTHOR]

Photomodulation of two-photon excited fluorescence has been effectively controlled in 1-pyrene-based dithienylethenes. The specific molecular design enables photocommutation of two-photon excited fluorescence during sequential one- and two-photon excitations without interference nor destructive optical readout. Theoretical studies have concluded that the internal functionalization of pyrene is responsible for the photoactivity in these systems. Photophysical studies indicate that the presence of a second laterally introduced pyrene is crucial for maintaining high photocommutation contrast and durability. [Display omitted] • Synthesis of new unsymmetrical pyrene-based DTE compound. • Photo-control of two-photon excitation signal. • Photo-switchable, all-optical, non-destructive molecular system. • Influence of the position of the pyrenes on the photophysical behavior. • Photo-modulation upon alternation of two- and one-photon excitation. [ABSTRACT FROM AUTHOR]

A series of novel fluorophores were obtained based on azolo[1,5- a ]pyrimidines (APs) through multicomponent and oxidation reactions, starting from aminoazoles, morpholineacrylonitrile, and 4-(dimethylamino)benzaldehyde. Photophysical studies, as well as DFT calculations have been performed for the obtained APs. Aggregation-induced emission (AIE) phenomenon was demonstrated for fluorophores containing an aromatic substituent at the C-2 position of the azolo[1,5- a ]pyrimidine core. It was observed that fluorophore 6d exhibited reversible mechanochromic luminescence. Two-photon absorption cross-section was measured for all obtained APs. In addition, cell staining resulted in identical intracellular distribution under excitation at 405 nm, 488 nm and 561 nm. [Display omitted] • A novel fluorescent dyes based on azolo[1,5- a ]pyrimidines (AP) have been synthesized. • These dyes exhibits solvatochromic emissions and aggregation-induced emission (AIE). • One of the representatives of azolopyrimdines exhibited reversible mechanochromic luminescence behavior. • Two-photon absorption was measured, and cell staining revealed identical intracellular distribution. • DFT calculations of HOMO/LUMO levels in molecules azolopyrimidines were undertaken. [ABSTRACT FROM AUTHOR]

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. HFZA combines the advantages of TP excitation and deep red emission with a high depth of penetration into biological tissues, allowing real-time monitoring of AChE activity in a C. elegans PD model as well as a mouse model of depression, and 3D imaging of tissue sections. [Display omitted] • A two-photon red fluorescent probe HFZA for imaging AChE was successfully created. • HFZA showed excellent specificity for AChE via computational docking over various interfering biological species. • High tissue penetration for in vivo imaging was achieved due to its two-photon absorption. • HFZA was able to detect AChE activity in both a C. elegans model of Parkinson's disease and a mouse model of depression. [ABSTRACT FROM AUTHOR]

Rydberg atoms possess highly excited valence electrons that are far away from atomic cations. Compared with ground states, Rydberg states are excited states with a high principal quantum number n that exhibit large electric dipole moments and have a variety of applications in quantum information processing. In this communication, we report the measurement of the 6Li Rydberg excitation spectrum by ladder-type electromagnetically induced transparency (EIT) in a vapor cell. The 2p→ns/nd EIT spectra were recorded by sweeping the frequency of an ultraviolet Rydberg pumping laser while keeping the probing laser resonant to the 2s→2p transition. All lasers were locked on an ultrastable optical Fabry-Pérot cavity and measured by an optical frequency comb. Our results provide valuable information to precisely determine quantum defects and enable novel experiments with Rydberg-dressed ultracold Fermi gases.

As a reactive hydrogen species, the hydrogen radical (H⋅) scarcely sees applications in tumor biological therapy due to the very limited bio‐friendly sources of H⋅. In this work, we report that TAF can act as an organic photosensitizer as well as an efficient photocatalytic H⋅ generator with reduced glutathione (GSH) as a fuel. The photoactivation of TAF leads to cell death in two ways including triple amplification of oxidative stress via ferroptosis‐apoptosis under normoxia and apoptosis through biological reductions under hypoxia. TAF presents excellent biosafety with ultrahigh photocytotoxicity index at an order of magnitude of 102–103 on both normoxic and hypoxic cells. The in vitro data suggest that H⋅ therapy is promising to overcome the challenge of tumor hypoxia at low doses of both photocatalyst and light. In addition, the capability of near‐infrared two‐photon excitation would benefit broad biological applications. [ABSTRACT FROM AUTHOR]

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study, we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong red-shifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released derivative of dicyanomethylene-4H-pyran (DCM-NH2) fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence, and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two-photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes. [ABSTRACT FROM AUTHOR]

Photoremovable protecting groups (PPGs) releasable by two‐photon excitation (2PE) provide spatio‐temporal control over the photoactivation of biological effectors to study biological systems. We synthesized four derivatives of the (8‐cyano‐7‐hydroxyquinolin‐2‐yl)methyl (CyHQ) chromophore by functionalization of position C4 to generate four different derivatives (MeO‐CyHQ, Mor‐CyHQ, pTol‐CyHQ, and TMP‐CyHQ) for studying the release of tertiary amines via 2PE. Sulpiride, an anti‐dopaminergic drug, was selected as a model substrate. All probes had excellent properties for use in biological settings, including high quantum yield (Φu), hydrolytic stability, and good aqueous solubility in simulated physiological buffer. The TMP‐CyHQ probe enhanced the two‐photon uncaging action cross‐section (δu) 8‐fold (2.64 GM) compared to the parent CyHQ‐sulpiride. The optimized PPG was used to mediate the photoactivation of various biological effectors containing the tertiary amine functionality (tamoxifen, 4‐hydroxytamoxifen, yohimbine) using 2PE. All constructs showed excellent efficiency with δu ranging from 1.21 to 2.42 GM and moderate to excellent yields of tertiary amines released. [ABSTRACT FROM AUTHOR]

Background: In addition to their great optical properties, nanodiamonds (NDs) have recently proved useful for two-photon-excited photodynamic therapy (TPE-PDT) applications. Indeed, they are able to produce reactive oxygen species (ROS) directly upon two-photon excitation but not with one-photon excitation; Methods: Fluorescent NDs (FNDs) with a 100 nm diameter and detonation NDs (DNDs) of 30 nm were compared. In order to use the gems for cancer-cell theranostics, they were encapsulated in a bis(triethoxysilyl)ethylene-based (ENE) periodic mesoporous organosilica (PMO) shell, and the surface of the formed nanoparticles (NPs) was modified by the direct grafting of polyethylene glycol (PEG) and amino groups using PEG-hexyltriethoxysilane and aminoundecyltriethoxysilane during the sol–gel process. The NPs' phototoxicity and interaction with MDA-MB-231 breast cancer cells were evaluated afterwards; Results: Transmission electronic microscopy images showed the formation of core–shell NPs. Infrared spectra and zeta-potential measurements confirmed the grafting of PEG and NH2 groups. The encapsulation of the NDs allowed for the imaging of cancer cells with NDs and for the performance of TPE-PDT of MDA-MB-231 cancer cells with significant mortality. Conclusions: Multifunctional ND@PMO core–shell nanosystems were successfully prepared. The NPs demonstrated high biocompatibility and TPE-PDT efficiency in vitro in the cancer cell model. Such systems hold good potential for two-photon-excited PDT applications. [ABSTRACT FROM AUTHOR]

Plant cells have a unique organization of microtubules during mitosis and cytokinesis. However, it is difficult to observe cell division in plant cells because of the distortion of images caused by thick samples. We addressed this issue by two-photon excitation coupled with spinning-disk confocal microscopy. Herein, we describe the method for observing microtubules and nuclei during cell division in tobacco BY-2 cells by two-photon spinning-disk confocal microscopy. The protocol includes the transformation of BY-2 cells with a plasmid for the expression of tubulin and histone markers, preparation of cells for microscopy, and operation of the two-photon spinning-disk microscope., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Precision-based drug delivery via remote triggering is fast becoming an attractive therapeutic design and is highly useful in complicated clinical situations that may require accurate site-delivery of drug while reducing the risk of collateral damage to surrounding healthy tissue. Of the many strategies available to achieve these desirable effects, silica/gold nano-assemblies offers a practical means to achieving these aims. Herein, as a proof-of-concept, a silica nanocapsule passivated with a gold outer nanoshell had been fabricated to deliver Doxorubicin, and this nano-assembly can be remotely triggered via two-photon excitation (TPE), even under in vivo setting. A polyethylene glycol (PEG) layer as well as AS1411 DNA aptamer had also been grafted to the surface to improve homing specificity toward MDA-MB-231 breast cancer tissue. The assembly of silica/gold nanocapsules was characterized via TEM, FTIR, and UV-Vis to validate the the nanoconstruct. Upon TPE irradiation, a higher expression level of Annexin V and Caspase-3 was observed in both in vitro and in vivo animal models. A significant reduction in tumor size on mice model was noticed after 21 days, and these results had suggested a viable nano-sized design serving as remotely triggered drug release platform based on current well-established silica nanoparticulate methodologies.

Much effort has been devoted to the generation of fluorescent probes by synthetic approaches. In this study, we developed a facile strategy to construct far‐red fluorescent probes based on through‐space charge transfer within complexes of acceptors and donors and their "twist+twist" interactions. Owing to their rare two‐photon excitation property, the complexes could be used for in vivo imaging of the mouse cerebrovascular system. [ABSTRACT FROM AUTHOR]

Abstract Developing novel photosensitizers for deep tissue imaging and efficient photodynamic therapy (PDT) remains a challenge because of the poor water solubility, low reactive oxygen species (ROS) generation efficiency, serve dark cytotoxicity, and weak absorption in the NIR region of conventional photosensitizers. Herein, cyclometalated iridium (III) complexes (Ir) with aggregation-induced emission (AIE) feature, high photoinduced ROS generation efficiency, two-photon excitation, and mitochondria-targeting capability were designed and further encapsulated into biocompatible nanoparticles (NPs). The Ir-NPs can be used to disturb redox homeostasis in vitro, result in mitochondrial dysfunction and cell apoptosis. Importantly, in vivo experiments demonstrated that the Ir-NPs presented obviously tumor-targeting ability, excellent antitumor effect, and low systematic dark-toxicity. Moreover, the Ir-NPs could serve as a two-photon imaging agent for deep tissue bioimaging with a penetration depth of up to 300 μm. This work presents a promising strategy for designing a clinical application of multifunctional Ir-NPs toward bioimaging and PDT.

We report on two-photon excited fluorescence interference patterns produced by spatial laser beam shaping. Thereto, a focused spatially modulated laser beam component is overlapped with an unfocused Gaussian beam component, and both are directed on a cuvette filled with dye. The phase retardance between the two common path laser components is precisely adjusted by a 2D liquid crystal modulator. With this method it is possible to generate various fluorescence patterns which exhibit sharp features and a high contrast due to constructive and destructive interference of a two-photon excitation process. The developed spatial shaping interference technique with two-photon excitations has a high potential for optical and biophotonic imaging applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • SHE-2N provides quick and stably stable labeling of plasma membrane. • No cytotoxicity detected for SHE-2N at concentrations used for imaging. • Suitable for STED nanoscopy and two-photon excitation microscopy. • SHE-2N offers high photostability. • Lipid composition does not affect emission or fluorescence lifetime of SHE-2N. Plasma membranes are vital biological structures, serving as protective barriers and participating in various cellular processes. In the field of super-resolution optical microscopy, stimulated emission depletion (STED) nanoscopy has emerged as a powerful method for investigating plasma membrane-related phenomena. However, many applications of STED microscopy are critically restricted by the limited availability of suitable fluorescent probes. This paper reports on the development of two amphiphilic membrane probes, SHE-2H and SHE-2 N , specially designed for STED nanoscopy. SHE-2 N , in particular, demonstrates quick and stable plasma membrane labelling with negligible intracellular redistribution. Both probes exhibit outstanding photostability and resolution improvement in STED nanoscopy, and are also suited for two-photon excitation microscopy. Furthermore, microscopy experiments and cytotoxicity tests revealed no noticeable cytotoxicity of probe SHE-2 N at concentration used for fluorescence imaging. Spectral analysis and fluorescence lifetime measurements conducted on probe SHE-2 N using giant unilamellar vesicles, revealed that emission spectra and fluorescence lifetimes exhibited minimal sensitivity to lipid composition variations. These novel probes significantly augment the arsenal of tools available for high-resolution plasma membrane research, enabling a more profound exploration of cellular processes and dynamics. [ABSTRACT FROM AUTHOR]

Monitoring neural activity and associating neural dynamics with the anatomical connectome are required to understand how the brain works. Neural dynamics are measured by electrophysiology and optical imaging. Since the discovery of the two-photon excitation phenomenon, significant progress has been made in deep imaging for capturing neural activity from numerous neurons in vivo. The development of two-photon microscopy is aimed to image neural activity from a large and deep region with high spatial (x , y , and z) and temporal (t) resolutions at a high signal-to-noise ratio. Imaging deep regions along the optical axis (z -axis) is particularly challenging because heterogeneous biological tissues scatter and absorb light. Recent advances in the light focus modulation technology at high speeds in three dimensions (x , y , and z) have allowed multiplane two-photon imaging. z -Focus control by varifocal optical systems, such as ferroelectric liquid lenses, gradient refractive index lenses, and adaptive optical element systems, and multiplexing by time- and wavelength-division strategies have allowed to rapidly observe specimens at different focal depths. Herein, we overview the recent advances in multiplane functional imaging systems that enable four-dimensional (x , y , z , and t) analysis of neural dynamics, with a special emphasis on z -scanning mechanisms and multiplexing strategies. • Multiplane two-photon imaging allows 4D (x , y , z , and t) analysis of neural dynamics. • Multiplane imaging is achieved using fast z -focus scanning and/or multiplexing techniques. • Fast varifocal optical system without moving objective is used in z -focus scanning. • Multiplexing via time- or wavelength-division allows signal separation and acquisition. [ABSTRACT FROM AUTHOR]

We report a self-assisted optothermal trapping and patterning of gold nanorods (GNRs) on glass surfaces with a femtosecond laser. We show that GNRs are not only the trapping targets, but also can enhance the optothermal trapping of other particles. This trapping phenomenon is the net result of thermophoresis and a convective flow caused by localized heating. The heating is due to the conversion of absorbed photons into heat at GNR's longitudinal surface plasmon resonance (LSPR) wavelength. First, we investigated the optothermal trapping of GNRs at their LSPR wavelength on the glass surface with as low as 0.5 mW laser power. The trapping range was observed to be larger than a typical field of view, e.g. 210 µm  ×  210 µm here. Second, by adjusting the distance between the laser focus and the glass surface, ring patterns of GNRs on the glass surface were obtained. These patterns could be controlled by the laser power and the numerical aperture of the microscope objective. Moreover, we examined the spectral emission of GNRs under different trapping conditions using the spectral phasor approach to reveal the temperature and association status of GNRs. Our study will help understanding manipulation of flows in solution and in biological systems that can be applied in future investigations of GNR-induced heating and flows.

Facile tailoring of photosensitizers (PSs) with advanced and synergetic properties is highly expected to broaden and deepen photodynamic therapy (PDT) applications. Herein, a catalyst‐free thiol–yne click reaction was employed to develop the sulfur atom‐based PSs by using the in situ formed sulfur "heavy atom effect" to enhance the intersystem crossing (ISC), while such an effect can be remarkably magnified by the polymerization. The introduction of a tetraphenylpyrazine‐based aggregation‐induced emission (AIE) unit was also advantageous in PS design by suppressing their non‐radiative decay to facilitate the ISC in the aggregated state. Besides, the resulting sulfur atom electron donor, together with a double‐bond π bridge and AIE electron acceptor, created a donor‐π‐acceptor (D‐π‐A) molecular system with good two‐photon excitation properties. Combined with the high singlet oxygen generation efficiency, the fabricated polymer nanoparticles exhibited an excellent in vitro two‐photon‐excited PDT towards cancer cells, therefore possessing a huge potential for the deep‐tissue disease therapy. [ABSTRACT FROM AUTHOR]

Precision-based drug delivery via remote triggering is fast becoming an attractive therapeutic design and is highly useful in complicated clinical situations that may require accurate site-delivery of drug while reducing the risk of collateral damage to surrounding healthy tissue. Of the many strategies available to achieve these desirable effects, silica/gold nano-assemblies offers a practical means to achieving these aims. Herein, as a proof-of-concept, a silica nanocapsule passivated with a gold outer nanoshell had been fabricated to deliver Doxorubicin, and this nano-assembly can be remotely triggered via two-photon excitation (TPE), even under in vivo setting. A polyethylene glycol (PEG) layer as well as AS1411 DNA aptamer had also been grafted to the surface to improve homing specificity toward MDA-MB-231 breast cancer tissue. The assembly of silica/gold nanocapsules was characterized via TEM, FTIR, and UV-Vis to validate the the nanoconstruct. Upon TPE irradiation, a higher expression level of Annexin V and Caspase-3 was observed in both in vitro and in vivo animal models. A significant reduction in tumor size on mice model was noticed after 21 days, and these results had suggested a viable nano-sized design serving as remotely triggered drug release platform based on current well-established silica nanoparticulate methodologies. [ABSTRACT FROM AUTHOR]

Wen-Shuo Kuo, 1, 2 Chia-Yuan Chang, 3 Jui-Chang Liu, 4 Jian-Hua Chen, 5, 6,* Edmund Cheung So, 5– 7,* Ping-Ching Wu 8,* 1School of Chemistry and Materials Science, Nanjing University of Information Science and Technology, Nanjing, Jiangsu 210044, People’s Republic of China; 2Allergy & Clinical Immunology Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan Republic of China; 3Department of Mechanical Engineering, National Cheng Kung University, Tainan 701, Taiwan Republic of China; 4Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan 701, Taiwan Republic of China; 5Department of Anesthesia & Medicine Research, An Nan Hospital, China Medical University, Tainan 709, Taiwan Republic of China; 6Department of Anesthesia, China Medical University, Taichung 404, Taiwan Republic of China; 7Graduate Institute of Medical Sciences, Chang Jung Christian University, Tainan 711, Taiwan Republic of China; 8Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan Republic of China*These authors contributed equally to this workCorrespondence: Jian-Hua Chen; Ping-Ching Wu Email aptx4869jfk@gmail.com; wbcxyz@bme.ncku.edu.twBackground: Multidrug-resistant (MDR) bacterial strain is a serious medical problem. Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to many antibiotics and is often associated with several diseases such as arthritis, osteomyelitis, and endocarditis. The development of an alternative treatment for eliminating MDR bacteria such as MRSA has attracted a considerable amount of research attention. Moreover, the development of a material for highly efficient generation of reactive oxygen species (ROS) involving two-photon photodynamic therapy (PDT) is currently desirable.Materials and Methods: We present an example demonstrating that the use of water-soluble C 60(OH) 30 fullerenol with a 0.89 singlet oxygen quantum yield serving as a photosensitizer in PDT has the superior ability in effectively generating ROS.Results: It has ultra-low energy (228.80 nJ pixel− 1) and can perform 900 scans under two-photon excitation (TPE) in the near-infrared region (760 nm) to completely eliminate the MDR species. Furthermore, the favorable two-photon properties are absorption of approximately 760 nm in wavelength, absolute cross-section of approximately 1187.50 Göeppert–Mayer units, lifetime of 6.640 ns, ratio of radiative to nonradiative decay rates of approximately 0.053, and two-photon stability under TPE.Conclusion: This enabled water-soluble C 60(OH) 30 fullerenol to act as a promising two-photon photosensitizer proceeding with PDT to easily eliminate MDR species.Keywords: methicillin-resistant Staphylococcus aureus, two-photon photodynamic therapy, water-soluble fullerenol, two-photon excitation, near-infrared region

Abstract We successfully prepared water-soluble fullerenol [C60(OH)46] that exhibited a high singlet oxygen quantum yield and efficiently generated reactive oxygen species. Additionally, the water-soluble C60(OH)46 with a higher composition of exposed hydroxyl groups had superior two-photon stability and characteristics compared with that with a lower composition of such groups. Therefore, the prepared fullerenol can be an effective two-photon photosensitizer. The water-soluble C60(OH)46 had favorable two-photon properties. During two-photon photodynamic therapy, the water-soluble C60(OH)46 had substantial antimicrobial activity against Escherichia coli at an ultralow-energy level of 211.2 nJ pixel−1 with 800 scans and a photoexcited wavelength of 760 nm.

Dark exciton states show great potential in condensed matter physics and optoelectronics because of their long lifetime and rich distribution in band structures. Therefore, they can theoretically serve as efficient energy reservoirs, providing a platform for future applications. However, their optical-transition-forbidden nature severely limits their experimental exploration and hinders their current application. Here, we demonstrate a universal dark state nonlinear energy transfer (ET) mechanism in monolayer WS 2 /CsPbBr 3 van der Waals heterostructures under two-photon excitation, which successfully utilizes the enormous energy reserved in the dark exciton state of CsPbBr 3 to significantly improve the photoelectric performance of monolayer WS 2 . We first propose the scenario of resonant ET between the dark state of CsPbBr 3 and WS 2 , and then reveal that this is a typical Förster resonant ET and belongs to the 2D-2D category. Interestingly, the dark state ET in CsPbBr 3 is identified as a long-range donor-bridge-acceptor hopping mode, with a potential distance exceeding 200 nm. Finally, we successfully achieve nearly an order of magnitude enhancement in the near-infrared detection performance of monolayer WS 2 . Our results enrich the theory of dark exciton states and ET, and they provide a way of using dark exciton states for future practical applications.

Microfluidic droplet sorting systems facilitate automated selective micromanipulation of compartmentalized micro- and nano-entities in a fluidic stream. Current state-of-the-art droplet sorting systems mainly rely on fluorescence detection in the visible range with the drawback that pre-labeling steps are required. This limits the application range significantly, and there is a high demand for alternative, label-free methods. Therefore, we introduce time-resolved two-photon excitation (TPE) fluorescence detection with excitation at 532 nm as a detection technique in droplet microfluidics. This enables label-free in-droplet detection of small aromatic compounds that only absorb in a deep-UV spectral region. Applying time-correlated single-photon counting, compounds with similar emission spectra can be distinguished due to their fluorescence lifetimes. This information is then used to trigger downstream dielectrophoretic droplet sorting. In this proof-of-concept study, we developed a polydimethylsiloxane-fused silica (FS) hybrid chip that simultaneously provides a very high optical transparency in the deep-UV range and suitable surface properties for droplet microfluidics. The herein developed system incorporating a 532-nm picosecond laser, time-correlated single-photon counting (TCSPC), and a chip-integrated dielectrophoretic pulsed actuator was exemplarily applied to sort droplets containing serotonin or propranolol. Furthermore, yeast cells were screened using the presented platform to show its applicability to study cells based on their protein autofluorescence via TPE fluorescence lifetime at 532 nm. [ABSTRACT FROM AUTHOR]

[Display omitted] In view of the photothermal effect of polydopamine (PDA) nanoparticles and their internal D-π-D structures during assembly, the two-photon excited properties of PDA were studied toward the biomedical application. Further, the PDA molecules were coordinated with Mn2+ and the assembled nanoparticles were covered by cancer cell membranes, the complex system could be used directly for the treatment of cancer with photothermal and chemodynamic therapy. The two-photon excited PDA-Mn2+ nanoparticles were used for the photothermal therapy combined with chemodynamic therapy. The complexes were coated with cancer cell membranes in order to enhance the tumor homologous efficiency. Multi-modal bioimaging and anti-tumor detections were carried out both in vitro and in vivo. PDA nanoparticles were demonstrated to have both good two-photon excited fluorescence and photothermal efficiency. The assembled nanoparticles modified with Mn2+ and cancer cell membranes have an obvious targeting and synergetic anti-cancer efficiency. The system creates a simple way for a precise operation with multi-modal imaging function. [ABSTRACT FROM AUTHOR]

Melanoma is a type of highly malignant and metastatic skin cancer. In situ molecular imaging of endogenous levels of the melanoma biomarker tyrosinase (TYR) may decrease the likelihood of mortality. In this study, we proposed the weakly fluorescent probe 1-(4-(2-(4-(dicyanomethylene)-4H-chromen-2-yl)vinyl)phenyl)-3-(4-hydroxybenzyl)urea (DCM-HBU), which releases a strong red-shifted fluorescent signal after a TYR-mediated oxidation followed by hydrolysis of the urea linkage. The large Stokes shift of the dye is owed to the recovery of the intramolecular charge transfer (ICT) effect. The resulting probe derivate shows a highly ratiometric fluorescence output. Furthermore, the simultaneous excitation by two near-infrared (NIR) photons of the released derivative of dicyanomethylene-4H-pyran (DCM-NH2) fluorophore could avoid the usual drawbacks, such as cellular absorption, autofluorescence, and light scattering, due to an usually short wavelength of the excitation light on biological systems, resulting in images with deeper tissue penetration. In addition, the probe is useful for the quantitative sensing of TYR activity in vivo, as demonstrated in zebrafish larvae. This new ratiometric two-photon NIR fluorescent probe is expected to be useful for the accurate detection of TYR in complex biosystems at greater depths than other one-photon excited fluorescent probes.

Gold nanoclusters (AuNCs) have attracted extensive attention as light-emissive materials with unique advantages such as high photostability, large Stoke shifts and low toxicity. However, a better understanding of their solid-state photoluminescence properties is still needed. Herein, we investigated for the first time the intrinsic photoluminescence properties of lyophilized bovine serum albumin stabilized AuNCs (BSA-AuNCs) via fluorescence lifetime imaging microscopy (FLIM) studies performed under both one and two photon excitations (OPE and TPE) on individual microflakes, combined with fluorescence spectroscopic investigations. Both in solution and solid-state, the synthesized BSA-AuNCs exhibit photoluminescence in the first biological window with an absolute quantum yield of 6% and high photostability under continuous irradiation. Moreover, under both OPE and TPE conditions, solid BSA-AuNCs samples exhibited a low degree of photobleaching, while FLIM assays prove the homogeneous distribution of the photoluminescence signal inside the microflakes. Finally, we demonstrate the ability of BSA-AuNCs to perform as reliable bright and photostable contrast agents for the visualization of cancer tissue mimicking agarose-phantoms using FLIM approach under non-invasive TPE. Therefore, our results emphasize the great potential of the as synthesized BSA-AuNCs for ex vivo and in vivo non-invasive NIR imaging applications.

The optogenetic method allows us to manipulate cellular activity with high spatiotemporal resolution. The most widely used optogenetic tool is channelrhodopsin-2 that can be utilized to control neuronal activity at the cellular level. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses (spines) have been lacking. We recently developed a photoactivatable CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Combining two-photon excitation, we successfully activated photoactivatable CaMKII in single spines and induced long-term potentiation (LTP) in hippocampal neurons. The manipulation of LTP at the single spines (we call it “local optogenetics”) will find many applications in neuroscience and other fields. [ABSTRACT FROM AUTHOR]