Search

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 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]

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]

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]

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]

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]

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]

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]

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]

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]

Ru(II) complexes with weak ligand fields may undergo light-induced ligand dissociation, and the resulted Ru(II) aqua complexes may bind with biomolecules such as DNA, showing potential as photoactivated chemotherapy (PACT) agents. However, Ru(II) complexes with efficient PACT activity are still rare. Some Ru(II) complexes exhibit efficient photoinduced ligand dissociation but poor cytotoxicity. It is speculated that the low nuclear accumulation levels may account for their low PACT efficacy. In order to confirm this hypothesis, the almost noncytotoxic [Ru(7-OCH3-dppz)(4-OCH3-py)4](PF6)2 (Ru1) is loaded on nucleus-targeted C5N2 nanoparticles (NPs). Compared with the free Ru1, Ru1-C5N2 NPs exhibit significantly increased cellular uptake and nuclear accumulation. Therefore, Ru1-C5N2 NPs show efficient PACT activity toward various cancer cell lines (including cisplatin-resistant one) with half maximal inhibitory concentration (IC50) values of 0.18 × 10-6-0.29 × 10-6 m and phototoxicity index (IC50 dark/IC50 light) values above 137 under both normoxic and hypoxic conditions. Moreover, Ru1-C5N2 NPs also exhibit efficient PACT activity toward cisplatin-resistant 3D multicellular tumor spheroids upon two-photon irradiation (800 nm). The same strategy is also feasible to greatly improve the PACT activity of [Ru(7-OCH3-dppz)(py)4]2+, which itself only has a medium effect. The results may provide new sights for developing efficient Ru(II) PACT agents. [ABSTRACT FROM AUTHOR]

The organoplatinum(II) complex [Pt(C^N^N)(Cl)] (C^N^N=5,6‐diphenyl‐2,2′‐bipyridine, Pt1) can assemble into nanoaggregates via π–π stacking and complementary hydrogen bonds, rather than Pt–Pt interactions. Pt1 exhibits ratiometric dual emission, including rare blue emission (λem=445 nm) and assembly‐induced yellow emission (λem=573 nm), under one‐ and two‐photon excitation. Pt1 displays blue emission in cells with an intact membrane due to its low cellular uptake. In cells where the membrane is disrupted, uptake of the complex is increased and at higher concentrations yellow emission is observed. The ratio of yellow to blue emission shows a linear relationship to the loss of cell membrane integrity. Pt1 is, to our knowledge, the first example of an assembly‐induced two‐photon ratiometric dual emission organoplatinum complex. The excellent and unique characteristics of the complex enabled its use for the tracking of cell apoptosis, necrosis, and the inflammation process in zebrafish. [ABSTRACT FROM AUTHOR]

Pilot calculations on the frequency‐dependent nonlinear response property, viz. the electric dipole hyperpolarizability of atomic hydrogen under quantum plasma environment, have been performed using an external oscillatory electric field. Fourth‐order perturbation theory within a variational scheme is adopted to obtain the hyperpolarizability within and beyond normal dispersion region. Two‐photon absorption from the ground state is explicitly obtained from the pole positions of nonlinear response of the system and studied up to principal quantum number n = 4. Ground and perturbed wave functions of appropriate symmetries are represented by linear combination of Slater‐type orbitals. Exponential cosine‐screened Coulomb potential is used to simulate the quantum plasma environment. With respect to plasma strength, the nonlinear response properties are considerably enhanced. Results are compared with those under classical plasma environment represented by screened Coulomb potential. Departure from Coulomb potential results in lifting of the accidental degeneracy in the respective two‐photon excited states beyond n = 2. For free hydrogen atom, the transition energies and the radial density profiles of the respective two‐photon excited states match exactly with those obtained from analytical wave functions. [ABSTRACT FROM AUTHOR]

Two‐dimensional (2D) organic–inorganic hybrid perovskites (OIHPs), a natural multiple‐quantum‐well structure with quasi‐2D electronic properties, have recently emerged as a promising class of semiconducting materials for photovoltaic and optoelectronic applications. However, facile synthesis of high‐quality 2D OIHPs single crystals is still lacking. The layer dependence of the exciton binding energy of (C4H9NH3)2PbI4 (C4PI), a widely studied 2D OIHP, is still debated. Herein, a novel synthesis technique based on inverse temperature crystallization in a binary‐solvent system is used to prepare 2D OIHPs and a systematic study of excitonic states of the synthesized 2D OIHPs by two‐photon excitation (TPE) spectroscopy is conducted. The obtained TPE spectra indicate that the exciton binding energies are similar for C4PI nanosheets and bulk crystals with different number of layers, most likely due to the intrinsically weak interlayer coupling. Further, the dark excitonic 2p states of (C6H5(CH2)2NH3)2PbI4 (PEPI) and C4PI are also observed by TPE spectroscopy. The results provide a novel synthesis protocol and insight into exciton properties of 2D OIHPs. [ABSTRACT FROM AUTHOR]

The early detection of cancer cells and their visualization before and after surgery are essential for successful pre‐ and post‐operative disease management. Although fluorescence imaging is a sensitive and versatile tool that is finding increasing use in clinical applications, there is a lack of tumour‐targeting fluorophores. We report here a family of fluorescent Schiff base organotin dyes (1: Et2N−L‐SnPh2, 2: Et2N−L‐SnBu2, 3: MeO−L‐SnPh2, 4: MeO−L‐SnBu2, 5: HO−L‐SnPh2, and 6: HO−L‐SnBu2, where L=2‐hydroxybenzylidene‐4‐hydroxybenzhydrazine). In addition to one‐photon‐excited fluorescence, efficient two‐photon excitation was demonstrated in compounds 1–4. Two of the compounds (5 and 6), both with hydroxyl substituents, were capable of selective accumulation in HeLa cells, allowing differentiation from normal cells (periodontal ligament cells). Compounds 1 and 3 showed excellent cancer cell staining (HeLa) using two‐photon bioimaging, which is promising for biomedicine applications. [ABSTRACT FROM AUTHOR]

Singlet oxygen (1O2) detection remains an active area of research in the field of biological and biomedical science. In this work, we reported a novel Off‐On type FRET‐based fluorescent 1O2 nanoprobe comprising the energy‐donating conjugated polymer, poly(9,9‐dioctylfluorenyl‐2,7‐diyl) (PFO), and the energy‐accepting 1O2 indicator, 3‐diphenylisobenzofuran (DPBF). Prior to the reaction with 1O2, the nanoprobe was in Off‐state because fluorescence of PFO (λem=441 nm) was completely quenched by loaded DPBF; in the presence of 1O2, it switched to On‐state due to the depletion of DPBF. The as‐prepared 1O2 nanoprobe had a particle size of ∼59 nm, good biocompatibility, high specificity and sensitivity with a limit of detection of 353 nM. Importantly, the fluorescent nanoprobes were highly sensitive to 1O2 under two‐photon excitation (λex=800 nm). Using a multiphoton confocal microscope, generation of intracellular 1O2 during photodynamic therapy was sensitively detected by recording fluorescence intensity of the nanoprobes. [ABSTRACT FROM AUTHOR]

Abstract: Photoswitchable bioprobes enable bidirectional control of cell physiology with different wavelengths of light. Many current photoswitches use cytotoxic UV light and are limited by the need for constant illumination owing to thermal relaxation in the dark. Now a photoswitchable tetrafluoroazobenzene(4FAB)‐based ion channel antagonist has been developed that can be efficiently isomerized in two separate optical channels with visible light. Importantly, the metastable cis configuration showed very high stability in the dark over the course of days at room temperature. In neurons, the 4FAB antagonist reversibly blocks voltage‐gated ion channels with violet and green light. Furthermore, photoswitching could also be achieved with two‐photon excitation yielding high spatial resolution. 4FAB probes have the potential to enable long‐term biological studies where both ON and OFF states can be maintained in the absence of irradiation. [ABSTRACT FROM AUTHOR]

Summary: Stimulated emission depletion (STED) microscopy is a useful tool in investigation for super‐resolution realm. By silencing the peripheral fluorophores of the excited spot, leaving only the very centre zone vigorous for fluorescence, the effective point spread function (PSF) could be immensely squeezed and subcellular structures, such as organelles, become discernable. Nevertheless, because of the low cross‐section of stimulated emission and the short fluorescence lifetime, the depletion power density has to be extremely higher than the excitation power density and molecules are exposed in high risk of photobleaching. The existence of photobleaching greatly limits the research of STED in achieving higher resolution and more delicate imaging quality, as well as long‐term and dynamic observation. Since the first experimental implementation of STED microscopy, researchers have lift out variety of methods and techniques to alleviate the problem. This paper would present some researches via conventional methods which have been explored and utilised relatively thoroughly, such as fast scanning, time‐gating, two‐photon excitation (TPE), triplet relaxation (T‐Rex) and background suppression. Alternatively, several up‐to‐date techniques, especially adaptive illumination, would also be unveiled for discussion in this paper. The contrast and discussion of these modalities would play an important role in ameliorating the research of STED microscopy. [ABSTRACT FROM AUTHOR]

Abstract: Photodynamic therapy (PDT) shows unique selectivity and irreversible destruction toward treated tissues or cells, but still has several problems in clinical practice. One is limited therapeutic efficiency, which is attributed to hypoxia in tumor sites. Another is the limited treatment depth because traditional photosensitizes are excited by short wavelength light (<700 nm). An assembled nano‐complex system composed of oxygen donor, two‐photon absorption (TPA) species, and photosensitizer (PS) was synthesized to address both problems. The photosensitizer is excited indirectly by two‐photon laser through intraparticle FRET mechanism for improving treatment depth. The oxygen donor, hemoglobin, can supply extra oxygen into tumor location through targeting effect for enhanced PDT efficiency. The mechanism and PDT effect were verified through both in vitro and in vivo experiments. The simple system is promising to promote two‐photon PDT for clinical applications. [ABSTRACT FROM AUTHOR]

Abstract: Perovskites have emerged as a class of cutting‐edge photovoltaic and light‐emitting materials. However, poor stability due to high moisture sensitivity and undesirable blinking severely limits their further application. Here, to solve these problems without destroying optoelectronic performance, a simple process for the fabrication of nonblinking CsPbBr3 quantum dots (QDs) is investigated. By embedding CsPbBr3 QDs into waterless silica spheres, the blinking of QDs can be strikingly suppressed, with an effective improvement of the moisture resistance and enhanced photostability. The silica sphere can also prevent anion exchange of different halide elements between perovskite QDs. Ultrastable amplified spontaneous emission (ASE) from QDs/SiO2 with no degradation for at least 12 h is observed under continuous laser irradiation (4 × 107 continuous intense laser shots), with almost no ASE degradation evident after 60 d of storage under ambient conditions. Most notably, the ASE threshold (Pth) of CsPbBr3 QDs is decreased by 50% and the relative efficiency increased by 388%. The perovskite QDs coated by the waterless SiO2 shell provide a novel platform for realizing perovskite nanomaterials with improved operational stability, nonblinking properties, and enhanced emission all at the same time, which is especially attractive for photovoltaic and light‐emitting device applications. [ABSTRACT FROM AUTHOR]

Photodynamic therapy (PDT) is a noninvasive protocol for the treatment of various cancers and nonmalignant diseases. Light, oxygen, and photosensitizer (PS) are the essential three elements in a typical PDT process. Currently, there are two major barriers limiting the further development of PDT. One issue is limited tissue penetration, and the other is the lack of high-performance PSs. Therefore, the newly emerging two-photon excited PDT (2PE-PDT) has attracted considerable attention in recent years due to its advantages such as a higher spatial resolution and a greater penetration depth. In this review, focus is on (i) the principle of 2PE-PDT, (ii) the progression of PSs for 2PE-PDT, and (iii) the potential indications and future directions in this field. [ABSTRACT FROM AUTHOR]

Luminescent temperature sensing in biological windows is uniquely superior compared to traditional temperature sensing methods because the scattering and absorption by skin, tissue and blood is minimal. In this study, the two-photon luminescence from dyes loaded into a metal-organic framework was utilised to achieve physiological temperature sensing within two biological windows. A composite was prepared that can be excited at 1064 nm to emit at 650 nm, well within the two biological windows. The two-photon luminescence intensity at 650 nm has a linear relationship with temperature over the range 20 to 60°C. In addition, the stability and quantum efficiency were significantly improved after loading into the MOF and, with high temperature resolution, excellent repeatability and low biological toxicity, measurements with this composite has great potential to be applied to physiological temperature sensing in cells or tissues. [ABSTRACT FROM AUTHOR]

A strongly fluorescent organic semiconducting polymer doped with a highly temperature dependent fluorescent europium(III) complex is converted into a nanosized material that is capable of optically sensing temperature ( T) in the range from 0 to 50 °C via two-photon excitation at 720 nm. The nanosensors are prepared from a blue-fluorescent polyfluorene that acts as both a light-harvesting antenna (to capture two-photon energy) and an energy donor in a fluorescence resonance energy transfer (FRET) system. The photonic energy absorbed by the polymer is transferred to the T-sensitive red-luminescent europium complex contained in the nanoparticles. The close spatial proximity of the donor and the acceptor warrants efficient FRET. A poly(ethylene glycol)- co-poly(propylene oxide) block copolymer is also added to render the particles biocompatible. It is shown that T can be calculated from a) the intensity of the luminescence of the europium complex, b) the ratio of the intensities of the red and blue luminescence, or c) the T-dependent luminescence lifetime of the Eu(III) complex. [ABSTRACT FROM AUTHOR]

Solution‐processed lead halide perovskites have shown good applicability in both solar cells and microlasers. Very recently, the nonlinear properties of perovskites have attracted considerable research attention. Second harmonic generation and two‐photon absorption have been successfully demonstrated. However, perovskite devices based on these nonlinear properties, such as micro‐ and nanolasers have thus far not been fabricated. Here we demonstrate two‐photon pumped microlasers from CH3NH3PbBr3 perovskite microwires. These CH3NH3PbBr3 perovskite microwires are synthesized through a one‐step solution precipitation method and dispersed on a glass substrate. Under optical excitation at 800 nm, two‐photon pumped lasing action with periodic peaks is successfully observed at around 546 nm. The obtained quality (Q) factors of the two‐photon pumped microlasers are around 682, and the corresponding thresholds are about 674 µJ cm‐2. Both the Q factors and thresholds are comparable to conventional whispering‐gallery modes in two‐dimensional polygon microplates. This work is the first demonstration of two‐photon pumped microlasers in CH3NH3PbBr3 perovskite microwires. We believe our finding will significantly expand the application of perovskites in low‐cost nonlinear optical devices, such as optical limiters, optical switches, and biomedical imaging devices. [ABSTRACT FROM AUTHOR]

Five cyclometalated iridium(III) complexes with 2-phenylimidazo[4,5- f][1,10]phenanthroline derivatives ( IrL1- IrL5) were synthesized and developed to image and track mitochondria in living cells under two-photon (750 nm) excitation, with two-photon absorption cross-sections of 48.8-65.5 GM at 750 nm. Confocal microscopy and inductive coupled plasma-mass spectrometry (ICP-MS) demonstrated that these complexes selectively accumulate in mitochondria within 5 min, without needing additional reagents for membrane permeabilization, or replacement of the culture medium. In addition, photobleaching experiments and luminescence measurements confirmed the photostability of these complexes under continuous laser irradiation and physiological pH resistance. Moreover, results using 3D multicellular spheroids demonstrate the proficiency of these two-photon luminescent complexes in deep penetration imaging. Two-photon excitation using such novel complexes of iridium(III) for exclusive visualization of mitochondria in living cells may substantially enhance practical applications of bioimaging and tracking. [ABSTRACT FROM AUTHOR]

Background In vivo multiphoton tomography ( MPT) of human skin has become a valuable tool for non-invasive examination of morphological and biophysical skin properties and their alterations. So far, skin changes after UVB irradiation were mainly evaluated clinically and histologically. The present study aimed at non-invasive imaging of histological changes during acute UVB irradiation by multiphoton laser tomography. Methods In 10 volunteers, five areas were irradiated once with an erythematous UVB dose. Multiphoton measurements were performed four times, i.e. before irradiation (baseline), and 24, 48 and 72 h after irradiation, respectively. The data were evaluated for changes of epidermal pleomorphy, spongiosis, pigmentation and thickness. Results The four parameters were altered significantly by acute UVB irradiation, i.e. epidermal pleomorphy, spongiosis, pigmentation and thickness increased within 72 h after irradiation. Conclusion Thus, the study has shown that typical epidermal changes induced by acute UVB irradiation can be evaluated by MPT. [ABSTRACT FROM AUTHOR]

ABSTRACT Mapping the activity of neuronal circuits with high resolution in the intact brain is a fundamental step toward understanding brain function. In the last several years, nonlinear microscopy combined with fluorescent activity reporters has become a crucial tool for achieving this goal. In this review article, we will highlight the principles underlying nonlinear microscopy and discuss its application to neuroscience, focusing on recent functional studies in the rodent neocortex in combination with genetically encoded calcium indicators. Microsc. Res. Tech. 77:492-501, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in the 1980s, which enabled imaging both fixed and living biological tissue with 3D precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to 2 years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning, and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. J. Comp. Neurol. 522:1708-1727, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

A therapy of cancer cells: Two‐photon‐triggered camptothecin delivery (see picture) with nanoimpellers was studied in MCF‐7 breast cancer cells. A fluorophore with a high two‐photon absorption cross‐section was first incorporated in the nanoimpellers. Fluorescence resonance energy transfer (FRET) from the fluorophore to the azobenzene moiety was demonstrated. [ABSTRACT FROM AUTHOR]

A new dinuclear RuII polypyridyl complex, [(bpy)2Ru(H2bpip)Ru(bpy)2]4+ ( RuH2bpip, bpy=2,2-bipyridine, H2bpip=2,6-pyridyl(imidazo[4,5-f][1,10]phenanthroline), was developed to act as a one- and two-photon luminescent probe for biological Cu2+ detection. This RuII complex shows a significant two-photon absorption cross section (400 GM) and displays a remarkable one- and two-photon luminescence switch in the presence of Cu2+ ions. Importantly, RuH2bpip can selectively recognise Cu2+ in aqueous media in the presence of other abundant cellular cations (such as Na+, K+, Mg2+, and Ca2+), trace metal ions in organisms (such as Zn2+, Ag+, Fe3+, Fe2+, Ni2+, Mn2+, and Co2+), prevalent toxic metal ions in the environment (such as Cd2+, Hg2+, and Cr3+), and amino acids, with high sensitivity (detection limit≤3.33×10−8 M) and a rapid response time (≤15 s). The biological applications of RuH2bpip were also evaluated and it was found to exhibit low cytotoxicity, good water solubility, and membrane permeability; RuH2bpip was, therefore, employed as a sensing probe for the detection of Cu2+ in living cells and zebrafish. [ABSTRACT FROM AUTHOR]

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue. [ABSTRACT FROM AUTHOR]

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the “renaissance” of light microscopy that started in the 1980s. The technique derives its advantages from the biologically “smooth” wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation–based assay system. [ABSTRACT FROM AUTHOR]

Two-photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP, EGFPand EYFPproduced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application. [ABSTRACT FROM AUTHOR]

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues. [ABSTRACT FROM AUTHOR]

Summary Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles. [ABSTRACT FROM AUTHOR]

Summary Two geometries of a novel two-photon fluorescence microscope incorporating single-mode fibre optics for the delivery of ultrashort-pulsed illumination to a remote sample are characterized. First, a 785 nm single-mode optical fibre is implemented in a scanning microscope, which demonstrates that an improvement in axial resolution is achieved due to the non-linear response of the fibre to intense ultrashort-pulsed light. Second, a 785 nm single-mode optical fibre coupler is adapted, in which case spectral broadening and blue shifting of the ultrashort-pulsed laser beam caused by the non-linear response of the fibre to ultrashort-pulsed illumination are experimentally characterized. An investigation into the impact of temporal broadening of the ultrashort-pulsed beam on the systems is also considered. The coupling efficiency of both geometries for various illumination wavelengths is also presented. The introduction of the fibre coupler to the system has significant advantages, including an improved optical sectioning effect and a reduction in the number of bulk optical components resulting in a low-cost, compact instrument. Sets of three-dimensional images of fluorescent polymer microspheres and biological material confirm these features. [ABSTRACT FROM AUTHOR]

A real-time two-photon fluorescence microscope with a microlens-array scanner using a regenerative amplifier is presented. The regenerative amplifier generates pulsed laser lights with extremely high peak power and produces brighter two-photon-induced fluorescence compared with that produced without the amplifier. Experimental results obtained from the observation of some biological samples with the proposed microscope are given. [ABSTRACT FROM AUTHOR]

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells. [ABSTRACT FROM AUTHOR]

Keywords: Singlet oxygen; nanoprobe; FRET; two-photon excitation; fluorescence quenching Under visible excitation or even NIR two-photon excitation, the initially non-fluorescent nanoprobe lit up in the presence of SP 1 sp O SB 2 sb . Singlet oxygen, nanoprobe, FRET, two-photon excitation, fluorescence quenching. [Extracted from the article]

Mitochondrial Imaging In their Full Paper on page 12000 ff., H. Chao and co ‐ workers report the synthesis of five IrIII complexes with two ‐ photon phosphorescence that were utilized for mitochondrial imaging and tracking in living cells. The complexes exhibit numerous advantages for two ‐ photon imaging, including rapid incubation time, appreciable photostability, excellent pH resistance, low imaging concentration, low cytotoxicity at the imaging concentration, sufficient two ‐ photon absorbing cross ‐ sections for imaging, and high resistance to decreases in the mitochondrial membrane potential, making them suitable for tracking mitochondrial morphological changes during the early stages of apoptosis. [ABSTRACT FROM AUTHOR]