Two-photon Laser Research Articles

Microstructured Cantilever Probe on Optical Fiber Tip for Microforce Sensor

Benefiting from the great advances of the femtosecond laser two-photon polymerization (TPP) technology, customized microcantilever probes can be accurately 3-dimensional (3D) manufactured at the nanoscale size and thus have exhibited considerable potentials in the fields of microforce, micro-vibration, and microforce sensors. In this work, a controllable microstructured cantilever probe on an optical fiber tip for microforce detection is demonstrated both theoretically and experimentally. The static performances of the probe are firstly investigated based on the finite element method (FEM), which provides the basis for the structural design. The proposed cantilever probe is then 3D printed by means of the TPP technology. The experimental results show that the elastic constant k of the proposed cantilever probe can be actively tuned from 2.46 N/m to 62.35 N/m. The force sensitivity is 2.5 nm/µN, the Q-factor is 368.93, and the detection limit is 57.43 nN. Moreover, the mechanical properties of the cantilever probe can be flexibly adjusted by the geometric configuration of the cantilever. Thus, it has an enormous potential for matching the mechanical properties of biological samples in the direct contact mode.

Evaluating the single-shot photodegradation of optically-transparent bulk polymers under two-photon Raman-resonant laser irradiation in nanosecond regime (multiplex CARS test)

Laser-induced photodegradation of bulk optically-transparent polymers was probed under laser irradiation at two-photon Raman resonance. The photodegradation effect was inspected for organic polymers poly-(methyl methacrylate) and polystyrene by means of single-shot multiplex coherent anti-Stokes Raman scattering (CARS) spectroscopy performed with nanosecond-pulsed laser sources. Under such conditions of photoirradiation in the visible spectral range, the laser pulse intensity threshold for photodegradation of these polymers was determined.

Measuring Rolling Friction at the Nanoscale.

Colloidal probe microscopy, a technique whereby a microparticle is affixed at the end of an atomic force microscopy (AFM) cantilever, plays a pivotal role in enabling the measurement of friction at the nanoscale and is of high relevance for applications and fundamental studies alike. However, in conventional experiments, the probe particle is immobilized onto the cantilever, thereby restricting its relative motion against a countersurface to pure sliding. Nonetheless, under many conditions of interest, such as during the processing of particle-based materials, particles are free to roll and slide past each other, calling for the development of techniques capable of measuring rolling friction alongside sliding friction. Here, we present a new methodology to measure lateral forces during rolling contacts based on the adaptation of colloidal probe microscopy. Using two-photon polymerization direct laser writing, we microfabricate holders that can capture microparticles, but allow for their free rotation. Once attached to an AFM cantilever, upon lateral scanning, the holders enable both sliding and rolling contacts between the captured particles and the substrate, depending on the interactions, while simultaneously giving access to normal and lateral force signals. Crucially, by producing particles with optically heterogeneous surfaces, we can accurately detect the presence of rotation during scanning. After introducing the workflow for the fabrication and use of the probes, we provide details on their calibration, investigate the effect of the materials used to fabricate them, and report data on rolling friction as a function of the surface roughness of the probe particles. We firmly believe that our methodology opens up new avenues for the characterization of rolling contacts at the nanoscale, aimed, for instance, at engineering particle surface properties and characterizing functional coatings in terms of their rolling friction.

Ca2+ oscillation in vascular smooth muscle cells control myogenic spontaneous vasomotion and counteract post-ischemic no-reflow

Ischemic stroke produces the highest adult disability. Despite successful recanalization, no-reflow, or the futile restoration of the cerebral perfusion after ischemia, is a major cause of brain lesion expansion. However, the vascular mechanism underlying this hypoperfusion is largely unknown, and no approach is available to actively promote optimal reperfusion to treat no-reflow. Here, by combining two-photon laser scanning microscopy (2PLSM) and a mouse middle cerebral arteriolar occlusion (MCAO) model, we find myogenic vasomotion deficits correlated with post-ischemic cerebral circulation interruptions and no-reflow. Transient occlusion-induced transient loss of mitochondrial membrane potential (ΔΨm) permanently impairs mitochondria-endoplasmic reticulum (ER) contacts and abolish Ca2+ oscillation in smooth muscle cells (SMCs), the driving force of myogenic spontaneous vasomotion. Furthermore, tethering mitochondria and ER by specific overexpression of ME-Linker in SMCs restores cytosolic Ca2+ homeostasis, remotivates myogenic spontaneous vasomotion, achieves optimal reperfusion, and ameliorates neurological injury. Collectively, the maintaining of arteriolar myogenic vasomotion and mitochondria-ER contacts in SMCs, are of critical importance in preventing post-ischemic no-reflow.

Behavioral dynamics of neuroprotective macrophage polarization in neuropathic pain observed by GHz femtosecond laser two-photon excitation microscopy.

Macrophage polarization in neurotoxic (M1) or neuroprotective (M2) phenotypes is known to play a significant role in neuropathic pain, but its behavioral dynamics and underlying mechanism remain largely unknown. Two-photon excitation microscopy (2PEM) is a promising functional imaging tool for investigating the mechanism of cellular behavior, as using near-infrared excitation wavelengths is less subjected to light scattering. However, the higher-order photobleaching effect in 2PEM can seriously hamper its applications to long-term live-cell studies. Here, we demonstrate a GHz femtosecond (fs) 2PEM that enables hours-long live-cell imaging of macrophage behavior with reduced higher-order photobleaching effect-by leveraging the repetition rate of fs pulses according to the fluorescence lifetime of fluorophores. Using this new functional 2PEM platform, we measure the polarization characteristics of macrophages, especially the long-term cellular behavior in efferocytosis, unveiling the dynamic mechanism of neuroprotective macrophage polarization in neuropathic pain. These efforts can create new opportunities for understanding long-term cellular dynamic behavior in neuropathic pain, as well as other neurobiological problems, and thus dissecting the underlying complex pathogenesis.

Nitrogen atoms absolute density measurement using two-photon absorption laser induced fluorescence in reactive magnetron discharge for gallium nitride deposition

Low-pressure plasmas, in particular magnetron sputtering discharges, are increasingly used for the deposition of wideband gap semiconductor nitrides films (e.g., GaN or AlN) considering many benefits they exhibit with respect to conventional chemical vapor deposition techniques. Plasma-based solutions enable the dissociation of N2 molecules into N-atoms under conditions that would not be possible with the thermal process. However, as the dissociation rate remains quite small due to the strong nitrogen triple bond, it is somewhat complicated to determine and correlate the N-atoms density in the gas phase with that of the grown film in low-pressure discharges. Therefore, ns-two-photon absorption laser induced fluorescence (TALIF) has been carried out to determine the absolute density of N-atoms as a function of the pressure (tens of Pa range) in a radio-frequency sputtering plasma reactor used for GaN deposition. The TALIF set-up has been optimized using a monochromator and adequate signal processing to enhance the detection limit, enabling the measurement of N-atoms density as low as 1011 cm−3 at 15 Pa. These measurements have been completed with electron density measurements performed in the same pressure range using microwave interferometry, thus providing quantitative data on both electron and N-atom densities that can be used for fundamental understanding, process optimization, and modeling of magnetron discharge intended for nitride semiconductor deposition.

Fabrication of Micro/Nano Dual Needle Structures with Morphological Gradient Based on Two-Photon Polymerization Laser Direct Writing with Proactive Focus Compensation

Micro/nano structures with morphological gradients possess unique physical properties and significant applications in various research domains. This study proposes a straightforward and precise method for fabricating micro/nano structures with morphological gradients utilizing single-voxel synchronous control and a nano-piezoelectric translation stage in a two-photon laser direct writing technique. To address the defocusing issue in large-scale fabrication, a methodology for laser focus dynamic proactive compensation was developed based on fluorescence image analysis, which can achieve high-precision compensation of laser focus within the entire range of the nano-piezoelectric translation stage. Subsequently, the fabrication of micro/nano dual needle structures with morphological gradients were implemented by employing different writing speeds and voxel positions. The minimum height of the tip in the dual needle structure is 80 nm, with a linewidth of 171 nm, and a dual needle total length reaching 200 μm. Based on SEM (scanning electron microscope) and AFM (atomic force microscope) characterization, the dual needle structures fabricated by the method proposed in this study exhibit high symmetry and nanoscale gradient accuracy. Additionally, the fabrication of hexagonal lattice periodic structures assembled from morphological gradient needle structures and the size gradient Archimedean spiral structures validate the capability of the single voxel-based fabrication and proactive focus compensation method for complex gradient structure fabrication.

Advancing Glucose Sensing Through Auto-Fluorescent Polymer Brushes: From Surface Design to Nano-Arrays.

Designing smart (bio)interfaces with the capability to sense and react to changes in local environments offers intriguing possibilities for new surface-based sensing devices and technologies. Polymer brushes make ideal materials to design such adaptive and responsive interfaces given their large variety of functional and structural possibilities as well as their outstanding abilities to respond to physical, chemical, and biological stimuli. Herein, a practical sensory interface for glucose detection based on auto-fluorescent polymer brushes decorated with phenylboronic acid (PBA) receptors is presented. The glucose-responsive luminescent surfaces, which are capable of translating conformational transitions triggered by pH variations and binding events into fluorescent readouts without the need for fluorescent dyes, are grown from both nanopatterned and non-patterned substrates. Two-photon laser scanning confocal microscopy and atomic force microscopy (AFM) analyses reveal the relationship between the brush conformation and glucose concentration and confirm that the phenylboronic acid functionalized brushes can bind glucose over a range of physiologically relevant concentrations in a reversible manner. The combination of auto-fluorescent polymer brushes with synthetic receptors presents a promising avenue for designing innovative and robust sensing systems, which are essential for various biomedical applications, among other uses.

Absolute atomic nitrogen density spatial mapping in three MHCD configurations

In this work, nanosecond two-photon absorption laser induced fluorescence (TALIF) is used to perform spatial mappings of the absolute density of nitrogen atoms generated in a micro-hollow cathode discharge (MHCD). The MHCD is operated in the normal regime, with a DC discharge current of 1.6 mA and the plasma is ignited in a 20% Ar/ 80% N2 gas mixture. A 1-inch diameter aluminum substrate, acting as a third electrode (second anode), is placed further away from the MHCD to emulate a deposition substrate. The spatial profile of the N atoms is measured in three MHCD configurations. First, we study a MHCD having the same pressure (50 mbar) on both sides of the anode/cathode electrodes and the N atoms simply diffuse in three dimensions from the MHCD. The recorded N atoms density profile in this case satisfies our expectations, i.e. the maximal density is found at the axis of the hole, close to the MHCD. However, when we introduce a pressure differential, thus creating a plasma jet, an unexpected N atoms distribution is measured with maximum densities away from the jet axis. This behavior cannot be simply explained by the TALIF measurements. Then, as a first simplified approach in this work, we turn our attention to the role of the gas flow pattern. Compressible gas flow simulations show a correlation between the jet width and the radial distribution of the N atoms at different axial distances from the gap. Finally, a DC positive voltage is applied to the third electrode (second anode), which ignites a micro cathode sustained discharge (MCSD). The presence of the pressure differential unveils two stable working regimes depending on the current repartition between the two anodes. The MCSD enables an homogenization of the density profile along the surface of the substrate, which is suitable for nitride deposition applications.

Anisotropic carrier dynamics and laser-fabricated luminescent patterns on oriented single-crystal perovskite wafers

Perovskite materials and their applications in optoelectronics have attracted intensive attentions in recent years. However, in-depth understanding about their anisotropic behavior in ultrafast carrier dynamics is still lacking. Here we explore the ultrafast dynamical evolution of photo-excited carriers and photoluminescence based on differently-oriented MAPbBr3 wafers. The distinct in-plane polarization of carrier relaxation dynamics of the (100), (110) and (111) wafers and their out-of-plane anisotropy in a picosecond time scale were found by femtosecond time- and polarization-resolved transient transmission measurements, indicating the relaxation process dominated by optical/acoustic phonon interaction is related to photoinduced transient structure rearrangements. Femtosecond laser two-photon fabricated patterns exhibit three orders of magnitude enhancement of emission due to the formation of tentacle-like microstructures. Such a ultrafast dynamic study carried on differently-oriented crystal wafers is believed to provide a deep insight about the photophysical process of perovskites and to be helpful for developing polarization-sensitive and ultrafast-response optoelectronic devices.

TOWARD CONTROLLED-RELEASE DRUG DELIVERY MICROCARRIERS ENABLED BY DIRECT LASER WRITING 3D PRINTING.

Controlled-release, and especially long-acting, drug delivery systems hold promise for improving treatments for numerous medical conditions. Previously, we reported an additive manufacturing or "three-dimensional (3D) printing" approach for fabricating liquid-core-shell-cap microcarriers comprising standard photoresists. Here we explore the potential to extend this strategy to achieve microcarriers comprising biodegradable materials as a new pathway to controlled-release drug delivery options. Specifically, we investigate the use of "Two-Photon Direct Laser Writing (DLW)" as a means to 3D print microcarriers composed of: (i) a bottle-shaped "shell" with an orifice, (ii) an aqueous liquid "core", and (iii) a biodegradable "cap". The cap, which is DLW-printed directly onto the shell's orifice, is designed to degrade over time in the body-e.g., with degradation time proportional to cap thickness-to ultimately facilitate release of the liquid core at desired time points. Fabrication results based on the use of a biodegradable poly(ethylene glycol) diacrylate (PEGDA) photomaterial for the cap revealed that shell designs incorporating microfluidic obstruction structures appeared to limit undesired entry of the liquid-phase PEGDA into the shell (i.e., directly preceding cap printing), thereby resulting in improved retention of the liquid core after completion of the cap printing process. These results mark an important first step toward evaluating the utility of the presented DLW 3D printing strategy for possible drug delivery applications.

FABRICATION OF MULTILUMEN MICROFLUIDIC TUBING FOR EX SITU DIRECT LASER WRITING.

Among the numerous additive manufacturing or "three-dimensional (3D) printing" techniques, two-photon Direct Laser Writing (DLW) is distinctively suited for applications that demand high geometric versatility with micron-to-submicron-scale feature resolutions. Recently, "ex situ DLW (esDLW)" has emerged as a powerful approach for printing 3D microfluidic structures directly atop meso/macroscale fluidic tubing that can be manipulated by hand; however, difficulties in creating custom esDLW-compatible multilumen tubing at such scales has hindered progress. To address this impediment, here we introduce a novel methodology for fabricating submillimeter multilumen tubing for esDLW 3D printing. Preliminary fabrication results demonstrate the utility of the presented strategy for resolving 743 μm-in-diameter tubing with three lumens-each with an inner diameter (ID) of 80 μm. Experimental results not only revealed independent flow of discrete fluorescently labelled fluids through each of the three lumens, but also effective esDLW-printing of a demonstrative 3D "MEMS" microstructure atop the tubing. These results suggest that the presented approach could offer a promising pathway to enable geometrically sophisticated microfluidic systems to be 3D printed with input and/or output ports fully sealed to multiple, distinct lumens of fluidic tubing for emerging applications in fields ranging from drug delivery and medical diagnostics to soft surgical robotics.

Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

Microinfarcts are common among the elderly and patients with microinfarcts are more vulnerable to another stroke. However, the impact of microinfarcts on recurrent stroke has yet to be fully understood. The purpose of this study was to explore the negative effects of microinfarcts on recurrent stroke. To achieve this, two-photon laser was used to induce microinfarcts, while photothrombotic stroke was induced on the opposite side. The results showed that microinfarcts led to trained immunity in microglia, which worsened the pro-inflammatory response and ischemic injury in the secondary photothrombotic stroke. Additionally, the study clarified the role of NLRP3 in microglial nuclei, indicating that it interacts with the MLL1 complex through NACHT domain and increases H3K4 methylation, which suggests that NLRP3 is critical in the formation of innate immune memory caused by microinfarcts. Furthermore, the knockout of NLRP3 in microglia alleviated the trained immunity and reduced the harmful effects of microinfarcts on recurrent stroke. This study emphasizes the detrimental effect of trained immunity on recurrent stroke and highlights the critical role of NLRP3 in mediating the formation of this memory, which may offer a potential therapeutic target for mitigating recurrent strokes.

In vivo label-free tissue histology through a microstructured imaging window.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.

Depopulation mechanisms of atomic hydrogen in the n = 3 level following two-photon excitation by a picosecond laser

One of the major constraints of measurements of atomic hydrogen densities using two-photon absorption laser induced fluorescence in most plasma and combustion environments is the determination of fluorescence decay times ( τfluo H ), especially when using nanosecond-lasers or slow acquisition systems. Therefore, it is necessary to identify the depopulation processes of the laser excited level in order to correctly estimate τfluo H . In this study, depopulation mechanisms of atomic hydrogen excited by two-photon absorption to the n = 3 level (H(n = 3)) have been investigated using a picosecond-laser excitation and acquisition of fluorescence by a streak camera, which allowed for direct measurement of τfluo H and hence, the atomic hydrogen densities, in an H2 microwave plasma operating in the pressure range 20–300 Pa. By combining these measurements with a detailed H(n = 3) collisional radiative depopulation model, it was found that full mixing amongst the H(n = 3) sub-levels occurs in our discharge conditions, even at a pressure as low as 20 Pa. Moreover, it is also seen that the Lyman β line is only partially trapped, as its escape factor Λ31 decreases from 0.94–0.98 down to 0.58–0.86 while the measured atomic hydrogen density rises from 8±5×1019m−3 to 9±6×1020m−3 . This means that the radiative decay rate of H(n = 3) atoms varies with pressure and the classical Stern–Volmer method used to determine the quenching cross-section of excited H(n = 3) in collisions with H2 molecules, σQHn=3/H2 , is not valid for our measurements. We used two different physics-based approaches, and show that the quenching cross-section σQHn=3/H2 lies in the range 90– 106×10−20m2 , which can be averaged as 98±8×10−20m2 . This substantially improved estimation of σQHn=3/H2 obtained in this work will be useful for the accurate estimation of H(n = 3) fluorescence decay times and therefore the atomic hydrogen densities.

Microscale manipulation of bond exchange reactions in photocurable vitrimers with a covalently attachable photoacid generator.

Vitrimers are polymer networks with covalent bonds that undergo reversible exchange reactions and rearrange their topology in response to an external stimulus. The temperature-dependent change in viscoelastic properties is conveniently adjusted by selected catalysts. In these thermo-activated systems, the lack in spatial control can be overcome by using photolatent catalysts. Herein, we advance this concept to locally manipulate bond exchange reactions on a single digit microscale level. For this, we synthetize a linkable non-ionic photoacid generator, which is covalently attached to a thiol-click photopolymer. UV induced deprotection of the photoacid yields a strong immobilized sulfonic acid species, which is able to efficiently catalyze transesterification reactions. Covalent attachment of the formed acid prevents migration/leaching processes and enables a precise tuning of material properties. As proof of concept, positive toned microstructures with a resolution of 5 μm are inscribed in thin films using direct two-photon absorption laser writing and subsequent depolymerization. In addition, the possibility to locally reprogram bulk material properties is demonstrated by performing a post-modification reaction with ethylene glycol and carboxylic acids. The Young's modulus is varied between 3.3 MPa and 11.9 MPa giving rise to the versatility of the newly introduced catalysts for creating light processable and transformable materials.

Reducing rubber-plastic friction in syringes through microstructured surface design and manufacturing

Plastic syringes often rely on silicone oil lubrication to reduce plunger-barrel friction, leading to potential issues like oil droplet release and drug aggregation. This study explored an alternative approach combining two-photon polymerization, laser machining, and microinjection molding to manufacture micro-dimpled structures for low friction. Plastic microdimples with high area density and low aspect ratio significantly reduced the coefficient of friction against rubber, while the dimple profile proved crucial in facilitating replication and demolding. The results of this study provide valuable insights into reducing friction between rubber and plastic, particularly in applications like syringes.

Alleviation of Post-sepsis Ischaemia by Drag-Reducing Polymers.

Sepsis, leading to septic shock and multiple organ dysfunction syndrome, is characterised by inflammation, coagulopathy, and microvascular dysfunction, the primary cause of in-hospital mortality. Novel approaches are needed to prevent the consequences of sepsis. We showed that nanomolar concentrations of intravascular blood-soluble drag-reducing polymers (DRPs) significantly improve microvascular perfusion and tissue oxygenation and protect neurons in rat brains after traumatic brain injury and haemorrhagic shock. The aim of this work was to determine whether DRPs-enhanced perfusion could alleviate sepsis-associated microvascular dysregulation in a mouse model of lipopolysaccharide (LPS)-induced sepsis. LPS (Salmonella Thyphosa, 10mg/kg, i.v.) was administered intravenously to induce acute sepsis in C57BL/6 J mice. DRPs (final concentration 5ppm in the blood) or saline was injected i.v. (10 mice/group) 1h after LPS injection to evaluate the efficacy of haemorheological modulation of microvascular dysregulation. In-vivo two-photon laser scanning microscopy was used to monitor cerebral (parietal cortex) and peripheral (ear) microcirculation (i.v. fluorescein isothiocyanate dextran) and tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence) at a baseline and during 4h after septic shock induction. Differences between groups were determined using a two-way analysis of variance for multiple comparisons with post hoc testing. The statistical significance was set at p<0.05. LPS-induced sepsis led to microvascular dysfunction and tissue hypoxia in the brain and peripheral tissue (ear). DRPs alleviated microthrombosis formation, microvascular dysfunction, and tissue hypoxia in the brain and peripheral tissue compared to the saline control group (p<0.05). Therefore, haemorheological modulation of blood flow by DRPs effectively improves systemic and peripheral circulation, reducing microthrombosis formation, microvascular dysfunction, and tissue hypoxia that can alleviate sepsis, shock, and multiple organ dysfunction syndrome.

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy.

In this study, two water-soluble red fluorescent protein (RFP) dimers, FP2R' and FP2R'', were synthesized by linking two phenothiazine-based RFP chromophore analogues through alkyl chains or alkoxy chains for hypoxic two-photon photodynamic therapy. RFP dimers are heavy-atom-free two-photon photosensitizers in which the intersystem crossing process is boosted by S and N heteroatoms. In terms of the aqueous solubility, the saturation concentration of FP2R'' was 3.5 mM, the emission wavelength was 677 nm, the singlet oxygen yield was 18%, and the two-photon absorption coefficient (β) was 2.1 × 10-11 cm W-1. Further, the RFP dimer FP2R'' showed excellent biocompatibility, negligible dark toxicity, and could produce 1O2 and O2˙- simultaneously. Under 460 nm illumination, the photosensitizer FP2R'' showed high phototoxicity with an IC50 value of 4.08 μM in an hypoxia environment, indicating that the photosensitizer FP2R'' has an excellent anti-hypoxia ability. In addition, the photosensitizer FP2R'' demonstrated a precise localization ability to lysosomes and its Pearson's colocalization coefficient was 0.94, which could guide the aggregation of photosensitizers in the lysosomes of tumor cells to effectively improve its photodynamic therapy (PDT) effect. In particular, when exposed to 800 nm two-photon excitation, FP2R'' effectively produced 1O2 and O2˙- in zebrafish and exhibited a bright two-photon fluorescence imaging capability. At the same time, the efficacy of two-photon photodynamic therapy mediated by the photosensitizer FP2R'' was verified in the tumor zebrafish model, and the growth of tumor cells in zebrafish was significantly inhibited under a two-photon laser irradiation. The water-soluble two-photon photosensitizer FP2R'' that was reasonably constructed in this study can be used as a high-efficiency hypoxic two-photon photosensitizer to inhibit deep tumor tissues.

Monoacylglycerol Lipase Inhibition Using ABX-1431 Attenuates Cerebral Ischaemia Early After Traumatic Brain Injury.

An early event in the pathology of traumatic brain injury (TBI) is a reduction in cerebral blood flow (CBF), which exacerbates secondary injury development and inhibits brain recovery. The endogenous cannabinoid system signalling (eCBs) might be critical in TBI recovery due to modulating synaptic activity and exerting neuroprotective and anti-inflammatory effects. In the brain, eCBs predominantly occur at cannabinoid receptor type 1 via the eCB 2-arachidonoylglycerol (2-AG). The aim of this work was to test the efficacy of potentiating 2-AG signalling by monoacylglycerol lipase (MAGL) inhibition using ABX-1431 immediately following TBI. Laser speckle contrast imaging (LSCI) was used to create a high-resolution map of regional cerebral blood flow (CBF) over the pericontusion cortical surface. In-vivo two-photon laser scanning microscopy (2PLSM) was used to monitor cerebral microcirculation (i.v. fluorescein isothiocyanate dextran, FITC) and mitochondrial respiration and brain tissue oxygen supply (nicotinamide adenine dinucleotide autofluorescence, NADH) during 4hours after CHI. After baseline imaging, male C57BL/6 J mice (10-12weeks, >28g) were subjected to a modified moderate Shohami weight-drop closed-head injury (CHI) followed by i.p. injection of ABX-1431 (5mg/kg) or vehicle 30 min after the insult (10 mice per group). Differences between groups and between time points were determined using two-way repeated measures (ANOVA) for multiple comparisons and post hoc testing with the statistical significance level set at p<0.05. Optical imaging revealed that CHI caused a decrease in regional CBF, arteriole diameters (vasospasm), and blood flow volume, leading to capillary microthrombosis and a reduction in capillary flow velocity. Compromised cerebral microcirculation led to the development of tissue hypoxia. ABX-1431 application, in a ~30-minute delay, mitigated the development of microvascular dysfunction, microthrombosis formation, and tissue hypoxia compared to the saline control group (p<0.05, starting 1hour after CHI). Therefore, MAGL inhibition by ABX-1431 attenuates cerebral ischaemia early after TBI. The observed 2-AG-mediated cerebrovascular relaxation might involve both a direct inhibition of smooth muscle contractility and a release of vasodilator mediator(s) from the endothelium.