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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]

Remotely triggered drug delivery using nanoparticles is an area of great interest for targeted therapy to fight cancer. In this work, we synthesized photoresponsive nanoparticles to remotely initiate the delivery of doxorubicin (DOX) to 3D cultured human breast cancer cells (MCF-7) via NIR two-photon excitation (TPE) using nitrogen-doped and surface passivated (PEG 200 ) carbon nanohybrid dots (CNDs). On-demand drug delivery relies on bio-compatible, photo-responsive nano-carriers with high quantum yields. A facile (5 min synthesis) one-pot hot plate method was used to synthesize functionalized and surface passivated carbon nanohybrid dots (CND-P) having 53% quantum yield (QY). Compared to CNDs prepared from citric acid (CND-C) and citric acid plus urea (CND-N), CND-P had QY enhanced by factors of 12.6 and 4.4, respectively. The up-converted emission intensity of CND-P was strengthened by a factor of 4.5 over that of CND-N from nitrogen doped CNDs for similar test conditions (wavelength, excitation power and concentration). The drug loading capacity of CND-P was measured to be 0.98 w/w with the ability to release DOX via two-photon excitation (TPE). Intense green luminescence was observed under both 360 and 780 nm lasers using single and two-photon excitations. The highly biocompatible CND-P showed 88% cell viability at concentrations as high as 1100 µg/mL. The combined chemo- and photothermal therapeutic effect of the DOX-loaded CND-P (CND-P@DOX) complex resulted in the death of 78% of the MCF-7 cells compared to 59% with DOX alone. [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]

Optogenetic neuronal network manipulation promises to unravel a long-standing mystery in neuroscience: how does microcircuit activity relate causally to behavioral and pathological states? The challenge to evoke spikes with high spatial and temporal complexity necessitates further joint development of light-delivery approaches and custom opsins. Two-photon (2P) light-targeting strategies demonstrated in-depth generation of action potentials in photosensitive neurons both in vitro and in vivo, but thus far lack the temporal precision necessary to induce precisely timed spiking events. Here, we show that efficient current integration enabled by 2P holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice. These results pave the way for optogenetic manipulation with the spatial and temporal sophistication necessary to mimic natural microcircuit activity. [ABSTRACT FROM AUTHOR]

C dots (CDs) have shown great potential in bioimaging and phototherapy. However, it is challenging to manipulate their fluorescent properties and therapeutic efficacy to satisfy the requirements for clinic applications. In this study, we prepared S, Se-codoped CDs via a hydrothermal method and demonstrated that the doping resulted in excitation wavelength-independent near-infrared (NIR) emissions of the CDs, with peaks at 731 and 820 nm. Significantly, the CDs exhibited a photothermal conversion efficiency of ~58.2%, which is the highest reported value for C nanostructures and is comparable to that of Au nanostructures. Moreover, the CDs had a large two-photon absorption cross section (~30,045 GM), which allowed NIR emissions and the photothermal conversion of the CDs through the two-photon excitation (TPE) mechanism. In vitro and in vivo tests suggested that CDs can function as new multifunctional phototheranostic agents for the TPE fluorescence imaging and photothermal therapy of cancer cells. [ABSTRACT FROM AUTHOR]

During the past two decades, great effort has been dedicated to two-photon excited colloidal quantum dots (QDs) because of their unique optical properties and significant applications. Under two-photon excitation, colloidal semiconductor QDs and carbon-family QDs show the nonlinear two-photon absorption (TPA) that is essential to the applications of two-photon microscopy and fluorescence up-conversion. For the first time, this review systematically summarizes the optical properties and potential applications of two-photon excited colloidal QDs with different categories. The optical properties of two-photon excited colloidal QDs include the typical nonlinear optical behavior, TPA, two-photon excited fluorescence (TPEF), time-resolved TPEF and TPEF lifetimes. The applications of two-photon excited colloidal QDs are wholly covered, including the cellular and in vivo imaging, chem./bio-detection, photodynamic therapy, controlled drug release, photocatalysis, photoelectric conversion, etc. This review comments the recent advances in the vibrant topic of colloidal QDs, and also highlights the optical phenomenon, related mechanisms and important applications. Finally, the probable challenges and perspectives in the forthcoming development of two-photon excited colloidal QDs are addressed. [ABSTRACT FROM AUTHOR]

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8 pg/mL (45 fM) with a wide linear range from 0.01 to 60 ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method. [ABSTRACT FROM AUTHOR]

Significance: Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are powerful tools for monitoring intracellular Ca 2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca 2+ 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 used 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 one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca 2+ imaging in hippocampal slices., Results: The Ca 2+ -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 Ca 2+ -dependent fluorescence increase is 15-fold and the apparent K d is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant., Conclusion: T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants., Competing Interests: Disclosures The authors declare no competing interests.

The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Photothermal therapy is a promising light-based medical treatment that relies on light absorption agents converting light irradiation into localized heat to destroy cancer cells or other diseased tissues. It is critical to enhance the therapeutic effects of cancer cell ablation for their practical applications. This study reports a high-performance combinational therapy for ablating cancer cells, including both photothermal therapy and chemotherapy to improve therapeutic efficiency. The prepared AuNR@mSiO 2 loading molecular Doxorubicin (Dox) assemblies were highlighted by merits of facile acquisition, great stability, easy endocytosis, and rapid drug release in addition to improved anticancer capability upon irradiation with a femtosecond pulsed near-infrared (NIR) laser, where AuNR@mSiO 2 nanoparticles afforded a high photothermal conversion efficiency of 31.7%. Two-photon excitation fluorescence imaging was introduced into confocal laser scanning microscope multichannel imaging to track the drug location and cell position in real time for monitoring the process of drug delivery in killing human cervical cancer HeLa cells and then to realize imaging-guiding cancer treatment. These nanoparticles exhibit widespread potential in photoresponsive utilizations including photothermal therapy, chemotherapy, one- and two-photon excited fluorescence imaging, and 3D fluorescence imaging and cancer treatment.

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

Organic inorganic hybrid perovskites have potential applications in solar cells, electroluminescent devices and radiation detection because of their unique optoelectronic properties. In this paper, the perovskites (C6H5CH2NH3)2PbBr4 and (C6H5CH2NH3)2PbI4 were synthesized by solvent evaporation. The crystal structure, morphology, absorption spectrum, laser power dependence of the photoluminescence (PL) intensity and lifetime were studied. The results showed that the perovskites (C6H5CH2NH3)2PbBr4 and (C6H5CH2NH3)2PbI4 display a layered stacking structure of organic and inorganic components. The absorption peaks are located at 392 nm (3.16 eV) and 516 nm (2.40 eV), respectively. It was observed that the PL intensity and photoluminescence quantum yield (PLQY) increases with increasing laser power, and that the PL lifetime decreases with increasing laser power, which is mainly due to the non-geminate recombination.

Nonlinear recording of stable fluorescent microstructures in PMMA films doped with photosensitive compound 2-(furan-2-yl)-3-(thiophen-2-carbonyl)-chromen-4-one is investigated. Energy and power density thresholds for recording stable fluorescent marks and for film destruction and photobleaching of the fluorescent marks by laser pulses were measured in wide spectral (from 530 to 620 nm) and temporal (from 80 to 7 ns) ranges. The evidences of the sequential two-step excitation mechanism for the studied dye are presented. It was shown that picosecond pulse duration range is preferable for two-photon data recording. Quasi-Gaussian and donut shaped fluorescent marks with almost the same cross-section were recorded by femtosecond pulses with rather small difference in energies. [ABSTRACT FROM AUTHOR]

Au@Ag nanoparticles (NPs) were recently found to display giant two-photon photoluminescence (2PPL) enhancement, with an enhancement factor up to 815 fold upon aggregate formation. Based on this finding, two-photon imaging of bacteria by Au@Ag NPs under near-infrared (NIR) femtosecond laser pulses was demonstrated in this study, as positively charged Au@Ag NPs can form aggregates on the negatively charged bacterial surface, yielding strong 2PPL emission. The aggregation-enhanced 2PPL of Au@Ag NPs stemmed from higher two-photon excitation efficiency, implying strong two-photon photothermal effects. Au@Ag NPs showed strong antibacterial activity (minimum inhibition concentration as low as 7.5 pM against Staphylococcus aureus ) and negligible toxicity to human dermal fibroblasts. Their bactericidal activity was further enhanced under NIR irradiation due to strong two-photon photothermal effects. Au@Ag NPs effectively removed 85% of the notorious bacterial biofilm within 4 min under NIR irradiation. These Au@Ag NPs can potentially be used as imaging and antibacterial agents. [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]

Singlet molecular oxygen, O 2 (a 1 Δ g ), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O 2 (a 1 Δ g ) in this regard is acknowledged, an understanding of the targets and mechanisms of O 2 (a 1 Δ g ) action is inadequate. Thus, methods that better facilitate studies of O 2 (a 1 Δ g ) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O 2 (a 1 Δ g ) can spawn the generation of other ROS ( e.g. , the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O 2 (a 1 Δ g ) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O 2 (a 1 Δ g ) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O 2 (a 1 Δ g ) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O 2 (a 1 Δ g ) at the expense of other ROS. We also comment on our recent efforts to monitor O 2 (a 1 Δ g ) in cells and to monitor the cell’s response to O 2 (a 1 Δ g ). [ABSTRACT FROM AUTHOR]

The paper is devoted to the theoretical investigation of two-photon excitation of atom in a discrete energy spectrum by ultrashort electromagnetic pulses of femto- and subfemtosecond ranges of durations. An analytical expression for the total probability of the process is derived. Numerical simulations are made for hydrogen and sodium atoms. It is shown that the total probability of the process is nonlinear function of pulse duration and character of this function depends strongly on the frequency detuning of pulse carrier frequency from two-photon resonance. [ABSTRACT FROM AUTHOR]

Reactive Oxygen Species, ROS, are small molecules ( e.g. , hydroxyl radical, superoxide radical anion, hydrogen peroxide, nitric oxide and singlet oxygen) that play key signaling roles in mammalian cells. They are generated as part of normal cell function, but also play roles in a cell’s response to perturbation ( e.g. , disease) and in many disease treatments ( e.g. , drugs). Although the importance of ROS is acknowledged, a general understanding of the mechanisms of ROS action and their biological effects is inadequate. Thus, new experimental methods that better facilitate studies of ROS behavior in mammalian cells are highly desired. In this feature article, we focus on one ROS in particular: singlet oxygen, O 2 (a 1 Δ g ). We summarize our recent efforts to selectively produce singlet oxygen in sub-cellular, spatially-resolved experiments performed on single mammalian cells. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to initially create a localized femtoliter volume of singlet oxygen, (2) protein-encapsulated photosensitizers that can be localized in cells using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments. We also provide a brief overview of our recent efforts to monitor singlet oxygen in cells ( e.g. , direct time-resolved optical detection, fluorescent probes) and to monitor the cell’s response to singlet oxygen ( e.g. , the use of rapid super-resolution microscopy). In all cases, we discuss the advantages and disadvantages of that particular approach/tool. [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]

The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the "Holy Grail" of cellular physiology. In an effort to obtain a minimally invasive approach to the mapping of intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 μm focused spot. Both methods utilized Ru coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium–sapphire Tsunami lasers (710–1050 nm).

Two-photon excitation fluorescence microscopy [two-photon microscopy (2PM)] is a robust technique for understanding physiological phenomena from the cellular to tissue level, attributable to the nonlinear excitation process induced by near-infrared ultrashort laser light pulses. Recently, we have been promoting the use of semiconductor lasers, adaptive optics, vector beams and nanomaterials to improve the observation depth or spatial resolution. The developed semiconductor-based laser light source successfully visualized the structure of the enhanced yellow fluorescent protein (EYFP)-expressing neurons at the hippocampal dentate gyrus without resecting the neocortex and neuronal activity in the hippocampal cornu ammonis (CA1) region in anesthetized mice at video rates. We also proposed using fluoropolymer nanosheets of 100-nm thickness for in vivo imaging and realized a wide field of view during anesthetized mouse brain imaging of 1-mm depth. Furthermore, the developed adaptive optical 2PM visualized single dendritic spines of EYFP-expressing neurons in cortical layer V of the secondary motor cortex, which had been difficult to observe due to the curvature of the brain surface. In addition, we combined 2PM and stimulated emission depletion microscopy to improve spatial resolution. This combined microscopy is noninvasive and has a superior spatial resolution, exceeding the diffraction limit of the conventional light. In this review, we describe our recent results and discuss the future of 2PM., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A new approach to calculate the effective Ln–Ln energy transfer rates in a doped crystal matrices was proposed and successfully applied to the YVO 4 :Yb 3+ ,Er 3+ upconversion system. This approach is based on the known set of Ln–Ln distances obtained from the crystal structure of the matrix and the probabilities of their occurrence. The non-radiative energy transfer rates were calculated in the intermediate coupling scheme taking into account the shielding of the 4f electrons. The required partial (that is, forced electric dipole-only, lacking the dynamic coupling contribution) Judd–Ofelt Ω λ parameters were calculated using the Simple Overlap Model (SOM). The spectral overlap factor F was estimated from optical absorption measurements. A simplified “one level-one manifold” energy level scheme was applied. The F 7 / 2 4 , H 11 / 2 2 and S 3 / 2 4 states of Er 3+ were treated as an effective level. The set of rate equations containing radiative, non-radiative and energy transfer processes was solved numerically using fourth-order Runge–Kutta method. Both continuous and pulsed excitation modes were simulated. The dependence of the upconversion intensity upon the pump power was found. The simulations resulted in good agreement with the experimental results and provided new insights into the dynamics of the upconversion process and a connection between the macroscopic properties and the microscopic description. [ABSTRACT FROM AUTHOR]

The photoluminescence (PL) from semiconductor quantum dots can show a “PL bright point”, that is the PL from as prepared quantum dots is maximum at a particular size. In this work we show that, for CdTe quantum dots, upconversion photoluminescence (UCPL) originating from nonlinear absorption shows a similar “UCPL bright point”. The PL and UCPL bright points occur at nearly the same size. The existence of a UCPL bright point has important implications for upconversion microscopy applications. [ABSTRACT FROM AUTHOR]

Weak up-converted fluorescence related to bacteriochlorophyll a was recorded from various detergent-isolated and membrane-embedded light-harvesting pigment-protein complexes as well as from the functional membranes of photosynthetic purple bacteria under continuous-wave infrared laser excitation at 1064 nm, far outside the optically allowed singlet absorption bands of the chromophore. The fluorescence increases linearly with the excitation power, distinguishing it from the previously observed two-photon excited fluorescence upon femtosecond pulse excitation. Possible mechanisms of this excitation are discussed. [ABSTRACT FROM AUTHOR]

A highly sensitive and selective two-photon sensing scheme for detection of cartap was developed by using Au@Ag bimetallic core-shell nanoparticles. Cartap was found to induce the aggregation of Au@Ag nanoparticles and up to 700-fold enhancement in two-photon photoluminescence. Huge enhancement in two-photon photoluminescence allows achieving a detection limit of as low as 0.0062 mg/kg, which is better than the conventional colorimetric methods. This two-photon sensing scheme has a broad dynamic range and displays excellent selectivity in detection of cartap against over other ten kinds of commonly used insecticides. [ABSTRACT FROM AUTHOR]

The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. [ABSTRACT FROM AUTHOR]

A multicolor fluorescent probe BNO for measuring intracellular pH is designed and synthesized, which is the first pH probe with a dual response mechanism combining an excited state intramolecular proton transfer (ESIPT) process and an intramolecular charge transfer (ICT) process. The probe shows negligible fluorescence at neutral and weak alkaline pH due to fluorescence quenching caused by ESIPT, and a sharp fluorescence enhancement in both acidic and strong alkaline environments. Meanwhile, it responds to pH changes with different emissions (green at acidic range, cyan at alkaline range). BNO is able to indicate pH variations in a wide range of 3.9–13.0. It is also capable of distinguishing intracellular pH values with high sensitivity. Furthermore, the probe exhibits favorable two-photon photophysics and high photostability, thus it is successfully applied to trace the dynamic change of intracellular pH with two-photon microscopy (TPM). The dual-mechanism strategy may be expanded to design other fluorescent probes. [ABSTRACT FROM AUTHOR]

Despite considerable advances in confocal microscopy and related technologies, advanced light microscopy techniques have been rarely used to study the microstructure of cheese matrices. Here, we demonstrate the potential of advanced microscopic techniques, namely fluorescence lifetime imaging (FLIM), confocal Raman spectroscopy and non-linear microscopy (two-photon excitation and second harmonic generation microscopy), to study localized physicochemical properties of cheese matrices. These methods allow for precise localization of individual chemical components and help to determine their spatial organization in three dimensions. We discuss both fluorescent labelling and label-free methods that provide valuable information about the localized environment. Overall, these new technologies will enable a greater understanding of the influence of manufacturing processes on cheese quality and consistency. [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]

With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited their imaging applications mostly to superficial tissues. To overcome these disadvantages, researchers have developed the graded-index (GRIN) probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. However, dynamic imaging based on GRIN lens has not been studied extensively. Here, this paper presented a fluorescence endoscopic imaging system based on GRIN lenses using one-photon and two-photon excitation. GRIN lenses with 1.15 mm diameter and 7.65 mm length were used in the system. The images were acquired by a compact laser scanning imaging system with a resonant galvo-mirror system to scan the laser beam and a photomultiplier tube (PMT) to detect fluorescence signals. Experimental results showed that this system using two-photon excitation could implement dynamic fluorescence microendoscopic imaging and monitor the movement of blood flow beneath the skin in anesthetized mice while producing images with higher contrast and signal to noise ratio (SNR) than those using one photon excitation. It would be a useful tool for studying biological processes of small animals or plants in vivo. [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]

Structural and optical characterization of crystalline Cu 2 O thin films obtained by thermal oxidation of Cu films at two different temperatures 800 °C and 900 °C are investigated in this work. X-ray diffraction measurements indicate that synthesized films consist of single Cu 2 O phase without any interstitial phase and show a nano-grain structure. Scanning Electron Microscopy observations indicate that the Cu 2 O films have a micro-scale roughness whereas High Resolution Transmission Electron Microscopy highlights that the nanocrystalline structure is formed by superposition of nearly spherical nanocrystals smaller than 30 nm. Photoluminescence spectra of these films exhibit at room temperature two well-resolved emission peaks at 1.34 eV due to defects energy levels and at 1.97 eV due to phonon-assisted recombination of the 1s orthoexciton in both film series. Emission characteristics depending on the laser power is deeply investigated to determine the origin of recorded emissions. Time-integrated spectra of the 1s orthoexciton emission reveals the presence of oxygen defects below the conduction band edge under non-resonant two-photon excitation using a wide range of excitations wavelengths. Optical absorption coefficients at room temperature are obtained from an accurate analysis of their transmission and reflection spectra, whereas the optical band gap energy is estimated at about 2.11 eV. Results obtained are of high relevance especially for potential applications in semiconductor devices such as solar cells, optical sources and detectors. [ABSTRACT FROM AUTHOR]

[Display omitted] • A two-photon photodynamic photosensitizer with mitochondrion-targeting and AIE characteristics was prepared. • The integration of Ir make 50 times enhancement of ROS production compared to its counterpart molecule. • TPABP-Ir@BSA NPs exhibits satisfactory biosafety. • A 90% tumor suppression was achieved by type-I&II synergetic photochemical reactions under hypoxic environment. In recent years, two-photon photodynamic therapy (TP-PDT) has received extensive attention in cancer treatment owing to its unique advantages of deep tissue penetration and high spatial–temporal controls. However, the high oxygen dependency of photosensitizers still remained as the major obstacle which undermined its overall therapeutic efficacy, especially in hypoxic-tolerant tumors. Exploration of new TP-PDT strategy with less-oxygen-dependency and subcellular organelle-targeting ability is an appealing yet seriously challenging task. In this work, a mitochondrion-targeting TP-PDT protocol based on type-I PSs with aggregation-induced emission (AIE) characteristics is proposed for the first time. Of the two synthesized AIE-active PSs, TPABP-Ir is demonstrated superior reactive oxygen species (ROS) production (both type I&II) owing to the doping of Ir(III). After encapsulation into bovine serum albumin (BSA) matrix, TPABP-Ir@BSA nanoparticles (Ir-NPs) are capable of realizing mitochondrion-targeting, high ROS production, and suppression of tumor growth under 880 nm femtosecond laser excitation. This study provides a novel treatment strategy which maximizes the PDT efficacy and offers a conceptual but practical paradigm for cancer treatment in translational nanomedicine. [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 10 2 -10 3 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., (© 2022 Wiley-VCH GmbH.)

We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-μm deep into the sample through the chitinous structures. The excitation power was kept below 10 mW(at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained. [ABSTRACT FROM AUTHOR]