Neurofibrillary tangle-bearing neurons have reduced risk of cell death in mice with Alzheimer's pathology.

A prevailing hypothesis is that neurofibrillary tangles play a causal role in driving cognitive decline in Alzheimer's disease (AD) because tangles correlate anatomically with areas that undergo neuronal loss. We used two-photon longitudinal imaging to directly test this hypothesis and observed the fate of individual neurons in two mouse models. At any time point, neurons without tangles died at >3 times the rate as neurons with tangles. Additionally, prior to dying, they became >20% more distant from neighboring neurons across imaging sessions. Similar microstructural changes were evident in a population of non-tangle-bearing neurons in Alzheimer's donor tissues. Together, these data suggest that nonfibrillar tau puts neurons at high risk of death, and surprisingly, the presence of a tangle reduces this risk. Moreover, cortical microstructure changes appear to be a better predictor of imminent cell death than tangle status is and a promising tool for identifying dying neurons in Alzheimer's.

Adapting and facilitating responses in mouse somatosensory cortex are dynamic and shaped by experience.

Sensory adaptation is the process whereby brain circuits adjust neuronal activity in response to redundant sensory stimuli. Although sensory adaptation has been extensively studied for individual neurons on timescales of tens of milliseconds to a few seconds, little is known about it over longer timescales or at the population level. We investigated population-level adaptation in the barrel field of the mouse somatosensory cortex (S1BF) using in vivo two-photon calcium imaging and Neuropixels recordings in awake mice. Among stimulus-responsive neurons, we found both adapting and facilitating neurons, which decreased or increased their firing, respectively, with repetitive whisker stimulation. The former outnumbered the latter by 2:1 in layers 2/3 and 4; hence, the overall population response of mouse S1BF was slightly adapting. We also discovered that population adaptation to one stimulus frequency (5 Hz) does not necessarily generalize to a different frequency (12.5 Hz). Moreover, responses of individual neurons to repeated rounds of stimulation over tens of minutes were strikingly heterogeneous and stochastic, such that their adapting or facilitating response profiles were not stable across time. Such representational drift was particularly striking when recording longitudinally across 8-9 days, as adaptation profiles of most whisker-responsive neurons changed drastically from one day to the next. Remarkably, repeated exposure to a familiar stimulus paradoxically shifted the population away from strong adaptation and toward facilitation. Thus, the adapting vs. facilitating response profile of S1BF neurons is not a fixed property of neurons but rather a highly dynamic feature that is shaped by sensory experience across days.

The Intermode Polariton Parametric Scattering Laser in a Strong Coupled Microcavity Via Two-Photon Absorption.

Exciton-polaritons, hybrid quasiparticles from the strong coupling of excitons and cavity photons in semiconductor microcavities, offer a platform for exploring quantum coherence and nonlinear optical properties. The unique polariton parametric scattering (PPS) laser is of interest for its potential in quantum technologies and nonlinear devices. However, direct resonant excitation of polaritons in strong-coupling microcavities is challenging. This study proposes an innovative two-photon absorption (TPA) pump mechanism to address this. We observe TPA-driven PPS lasing in a strongly coupled microcavity at room temperature. High K-value exciton injections promote coherent stimulated emission of polariton scattering through intermode channels. Angle-resolved spectra confirm a TPA process, showing evolution from pump-state to signal-state. Hanbury Brown-Twiss measurement of second-order correlation g2(τ) of signal state indicates a phase transition from a classical thermal state to a quantum coherent state. Theoretical modeling provides insights into the physical mechanisms of PPS. Our work advances nonlinear phenomena exploration in strongly coupled light-matter systems, contributing to quantum polaritonics and nonlinear optics.

Coumarin-Based Photodegradable Hydrogels Enable Two-Photon Subtractive Biofabrication at 300†mm s-1.

Spatiotemporally controlled two-photon photodegradation of hydrogels has gained increasing attention for high-precision subtractive tissue engineering. However, conventional photolabile hydrogels often have poor efficiency upon two-photon excitation in the near-infrared (NIR) region and thus require high laser dosage that may compromise cell activity. As a result, high-speed two-photon hydrogel erosion in the presence of cells remains challenging. Here we introduce the design and synthesis of efficient coumarin-based photodegradable hydrogels to overcome these limitations. A set of photolabile coumarin-functionalized polyethylene glycol linkers are synthesized through a Passerini multicomponent reaction. After mixing these linkers with thiolated hyaluronic acid, semi-synthetic photodegradable hydrogels are formed in situ via Michael addition crosslinking. The efficiency of photodegradation in these hydrogels is significantly higher than that in nitrobenzyl counterparts upon two-photon irradiation at 780 nm. A complex microfluidic network mimicking the bone microarchitecture is successfully fabricated in preformed coumarin hydrogels at high speeds of up to 300 mm s-1 and low laser dosage down to 10 mW. Further, we demonstrate fast two-photon printing of hollow microchannels inside a hydrogel to spatiotemporally direct cell migration in 3D. Collectively, these hydrogels may open new avenues for fast laser-guided tissue fabrication at high spatial resolution.

Clustered synapses develop in distinct dendritic domains in visual cortex before eye opening.

Synaptic inputs to cortical neurons are highly structured in adult sensory systems, such that neighboring synapses along dendrites are activated by similar stimuli. This organization of synaptic inputs, called synaptic clustering, is required for high-fidelity signal processing, and clustered synapses can already be observed before eye opening. However, how clustered inputs emerge during development is unknown. Here, we employed concurrent in vivo whole-cell patch-clamp and dendritic calcium imaging to map spontaneous synaptic inputs to dendrites of layer 2/3 neurons in the mouse primary visual cortex during the second postnatal week until eye opening. We found that the number of functional synapses and the frequency of transmission events increase several fold during this developmental period. At the beginning of the second postnatal week, synapses assemble specifically in confined dendritic segments, whereas other segments are devoid of synapses. By the end of the second postnatal week, just before eye opening, dendrites are almost entirely covered by domains of co-active synapses. Finally, co-activity with their neighbor synapses correlates with synaptic stabilization and potentiation. Thus, clustered synapses form in distinct functional domains presumably to equip dendrites with computational modules for high-capacity sensory processing when the eyes open.

Precise Serial Microregistration Enables Quantitative Microscopy Imaging Tracking of Human Skin Cells In Vivo.

We developed an automated microregistration method that enables repeated in vivo skin microscopy imaging of the same tissue microlocation and specific cells over a long period of days and weeks with unprecedented precision. Applying this method in conjunction with an in vivo multimodality multiphoton microscope, the behavior of human skin cells such as cell proliferation, melanin upward migration, blood flow dynamics, and epidermal thickness adaptation can be recorded over time, facilitating quantitative cellular dynamics analysis. We demonstrated the usefulness of this method in a skin biology study by successfully monitoring skin cellular responses for a period of two weeks following an acute exposure to ultraviolet light.

Identification of cortical arteries and veins in awake mice using two-photon microscopy.

Distinguishing arteries from veins in the cerebral cortex is critical for studying hemodynamics under pathophysiological conditions, which plays an important role in the diagnosis and treatment of various vessel-related diseases. However, due to the complexity of the cerebral vascular network, it is challenging to identify arteries and veins in vivo. Here, we demonstrate an artery-vein separation method that employs a combination of multiple scanning modes of two-photon microscopy and a custom-designed stereoscopic fixation device for mice. In this process, we propose a novel method for determining the line scanning direction, which allows us to determine the blood flow directions. The vasculature branches have been identified using an optimized z-stack scanning mode, followed by the separation of blood vessel types according to the directions of blood flow and branching patterns. Using this strategy, the penetrating arterioles and penetrating venules in awake mice could be accurately identified and the type of cerebral thrombus has been also successfully isolated without any empirical knowledge or algorithms. Our research presents a new, more accurate, and efficient method for cortical artery-vein separation in awake mice, providing a useful strategy for the application of two-photon microscopy in the study of cerebrovascular pathophysiology.

Advances in two-photon absorption photodynamic therapy of glioma based on porphyrin-based metal-organicframework composites.

Gliomas of the brain are characterised by high aggressiveness, high postoperative recurrence rate, high morbidity and mortality, posing a great challenge to clinical treatment. Traditional treatments include surgery, radiotherapy and chemotherapy; they also have significant associated side effects, leading to difficulties in tumour resection and recurrence. Photodynamic therapy has been shown to be a promising new strategy to help treat malignant tumours of the brain. It irradiates the tumour site at a specific wavelength to activate a photosensitiser, which selectively accumulates at the tumour site, triggering a photochemical reaction that destroys the tumour cells. It has the advantages of being minimally invasive, highly targeted and with few adverse reactions, and is expected to be well used in anti-tumour therapy. However, the therapeutic effect of traditional PDT is limited by the weak tissue penetration ability of photosensitiser, hypoxia and immunosuppression in the tumour microenvironment. This paper reviews the current research status on the therapeutic principle of photodynamic therapy in glioma and the mechanism of tumour cell injury, and also analyses the advantages and disadvantages of the current application in glioma treatment, and clarifies the analysis of ideas to improve the tissue penetration ability of photosensitizers. It aims to provide a feasible direction for the improvement of photodynamic therapy for glioma and a reference for the clinical treatment of deep brain tumours.

Sub-Micron Replication of Fused Silica Glass and Amorphous Metals for Tool-Based Manufacturing.

The growing importance of submicrometer-structured surfaces across a variety of different fields has driven progress in light manipulation, color diversity, water-repellency, and functional enhancements. To enable mass production, processes like hot-embossing (HE), roll-to-roll replication (R2R), and injection molding (IM) are essential due to their precision and material flexibility. However, these processes are tool-based manufacturing (TBM) techniques requiring metal molds, which are time-consuming and expensive to manufacture, as they mostly rely on galvanoforming using templates made via precision microlithography or two-photon-polymerization (2PP). In this work, a novel approach is demonstrated to replicate amorphous metals from fused silica glass, derived from additive manufacturing and structured using hot embossing and casting, enabling the fabrication of metal insets with features in the range of 300 nm and a surface roughness of below 10 nm. By partially crystallizing the amorphous metal, during the replication process, the insets gain a high hardness of up to 800 HV. The metal molds are successfully used in polymer injection molding using different polymers including polystyrene (PS) and polyethylene (PE) as well as glass nanocomposites. This work is of significant importance to the field as it provides a production method for the increasing demand for sub-micron-structured tooling in the area of polymer replication while substantially reducing their cost of production.

Monitoring correlates of SARS-CoV-2 infection in cell culture using a two-photon-active calcium-sensitive dye.

BACKGROUND: The organism-wide effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection are well studied, but little is known about the dynamics of how the infection spreads in time among or within cells due to the scarcity of suitable high-resolution experimental systems. It has been reported that SARS-CoV-2 infection pathways converge at calcium influx and subcellular calcium distribution changes. Imaging combined with a proper staining technique is an effective tool for studying subcellular calcium-related infection and replication mechanisms at such resolutions. METHODS: Using two-photon (2P) fluorescence imaging with our novel Ca-selective dye, automated image analysis and clustering analysis were applied to reveal titer and variant effects on SARS-CoV-2-infected Vero E6 cells. RESULTS: The application of a new calcium sensor molecule is shown, combined with a high-end 2P technique for imaging and identifying the patterns associated with cellular infection damage within cells. Vero E6 cells infected with SARS-CoV-2 variants, D614G or B.1.1.7, exhibit elevated cytosolic calcium levels, allowing infection monitoring by tracking the cellular changes in calcium level by the internalized calcium sensor. The imaging provides valuable information on how the level and intracellular distribution of calcium are perturbed during the infection. Moreover, two-photon calcium sensing allowed the distinction of infections by two studied viral variants via cluster analysis of the image parameters. This approach will facilitate the study of cellular correlates of infection and their quantification depending on viral variants and viral load. CONCLUSIONS: We propose a new two-photon microscopy-based method combined with a cell-internalized sensor to quantify the level of SARS-CoV-2 infection. We optimized the applied dye concentrations to not interfere with viral fusion and viral replication events. The presented method ensured the proper monitoring of viral infection, replication, and cell fate. It also enabled distinguishing intracellular details of cell damage, such as vacuole and apoptotic body formation. Using clustering analysis, 2P microscopy calcium fluorescence images were suitable to distinguish two different viral variants in cell cultures. Cellular harm levels read out by calcium imaging were quantitatively related to the initial viral multiplicity of infection numbers. Thus, 2P quantitative calcium imaging might be used as a correlate of infection or a correlate of activity in cellular antiviral studies.

Enhanced third-order nonlinear optical properties of ZnO@C-N composite microspheres.

We report the third-order nonlinear optical (NLO) properties of ZnO@C-N composite microspheres and pure ZnO which have been investigated with the Z-scan technique under continuous wave laser. ZnO@C-N composite microspheres have been hydrothermally synthesized at two different precursor concentrations to have structures at different impurity levels. Moreover, pure ZnO is prepared under the annealing process. The nonlinear optical absorption of samples was measured by using the open-aperture Z-scan technique and was evaluated relating to the two-photon absorption (TPA) mechanism. Moreover, both ZnO@C-N and ZnO microstructures exhibited a negative nonlinear refractive index (NLR) referring to the self-defocusing effect. The order of the (NLR) value, is about 10-10(cm2/W) and, the NLA coefficients of specimens are in the order of 10-5(cm/W). The NLA coefficient has a similar behavior as the NLR versus increasing incident intensity of the laser. The results show that the nonlinearity response of ZnO@C-N composites is higher than the pure ZnO and ZnO@C-N at higher precursor concentrations exhibits the maximum amount of NLA and NLR coefficients compared to other samples. This observation which is attributed to the change in optical and structural properties of material due to impurity presence, underscores the presence of impurity for engineering materials to improve the nonlinearity properties.

SomaSeg: A robust neuron identification framework for two-photon imaging video.

Accurate neuron identification is fundamental to the analysis of neuronal population dynamics and signal extraction in fluorescence videos. However, several factors such as severe imaging noise, out-of-focus neuropil contamination, and adjacent neuron overlap would impair the performance of neuron identification algorithms and lead to errors in neuron shape and calcium activity extraction, or ultimately compromise the reliability of analysis conclusions. Herein, to address these challenges, we developed a novel cascade framework - SomaSeg that combines Duffing denoising, neuropil contamination defogging and stacked instance differentiating. Compared with the state-of-the-art neuron identification methods, both simulation and actual experimental results demonstrate that SomaSeg framework is robust to noise, insensitive to out-of-focus contamination and effective in dealing with overlapping neurons in actual complex imaging scenarios, providing a widely applicable framework for two-photon video processing.

Population coding for figure-ground texture segregation in macaque V1 and V4.

Object recognition often involves the brain segregating objects from their surroundings. Neurophysiological studies of figure-ground texture segregation have yielded inconsistent results, particularly on whether V1 neurons can perform figure-ground texture segregation or just detect texture borders. To address this issue from a population perspective, we utilized two-photon calcium imaging to simultaneously record the responses of large samples of V1 and V4 neurons to figure-ground texture stimuli in awake, fixating macaques. The average response changes indicate that V1 neurons mainly detect texture borders, while V4 neurons are involved in figure-ground segregation. However, population analysis (SVM decoding of PCA-transformed neuronal responses) reveal that V1 neurons not only detect figure-ground borders, but also contribute to figure-ground texture segregation, although requiring substantially more principal components than V4 neurons to reach a 75 % decoding accuracy. Individually, V1/V4 neurons showing larger (negative/positive) figure-ground response differences contribute more to figure-ground segregation. But for V1 neurons, the contribution becomes significant only when many principal components are considered. We conclude that V1 neurons participate in figure-ground segregation primarily by defining the figure borders, and the poorly structured figure-ground information V1 neurons carry could be further utilized by V4 neurons to accomplish figure-ground segregation.

Neutrophil Migration Is Mediated by VLA-6 in the Inflamed Adipose Tissue.

Adipose tissue, well known for its endocrine function, plays an immunological role in the body. The inflamed adipose tissue under LPS-induced systemic inflammation is characterized by the dominance of pro-inflammatory immune cells, particularly neutrophils. Although migration of macrophages toward damaged or dead adipocytes to form a crown-like structure in inflamed adipose tissue has been revealed, the neutrophilic interaction with adipocytes or the extracellular matrix remains unknown. Here, we demonstrated the involvement of adhesion molecules, particularly integrin α6ß1, of neutrophils in adipocytes or the extracellular matrix of inflamed adipose tissue interaction. These results suggest that disrupting the adhesion between adipose tissue components and neutrophils may govern the accumulation of excessive neutrophils in inflamed tissues, a prerequisite in developing anti-inflammatory therapeutics by inhibiting inflammatory immune cells.

The NKCC1 inhibitor bumetanide restores cortical feedforward inhibition and lessens sensory hypersensitivity in early postnatal fragile X mice.

BACKGROUND: Exaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice. METHODS: We used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos. RESULTS: We demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons. CONCLUSIONS: This further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Triphenylamine Sensitized 8-Dimethylaminoquinoline: An Efficient Two-Photon Caging Group for Intracellular Delivery.

Triphenylamine-sensitized 8-dimethylaminoquinoline (TAQ) probes showed fair two-photon absorption and fragmentation cross sections in releasing kainate and GABA ligands. The water-soluble PEG and TEG-analogs allowed cell internalization and efficient light-gated liberation of the rhodamine reporter under UV and two-photon (NIR) irradiation conditions.

Integrity of neural extracellular matrix is required for microglia-mediated synaptic remodeling.

Microglia continuously remodel synapses, which are embedded in the extracellular matrix (ECM). However, the mechanisms, which govern this process remain elusive. To investigate the influence of the neural ECM in synaptic remodeling by microglia, we disrupted ECM integrity by injection of chondroitinase ABC (ChABC) into the retrosplenial cortex of healthy adult mice. Using in vivo two-photon microscopy we found that ChABC treatment increased microglial branching complexity and ECM phagocytic capacity and decreased spine elimination rate under basal conditions. Moreover, ECM attenuation largely prevented synaptic remodeling following synaptic stress induced by photodamage of single synaptic elements. These changes were associated with less stable and smaller microglial contacts at the synaptic damage sites, diminished deposition of calreticulin and complement proteins C1q and C3 at synapses and impaired expression of microglial CR3 receptor. Thus, our findings provide novel insights into the function of the neural ECM in deposition of complement proteins and synaptic remodeling by microglia.

Click Synthesis of Tweezer Dyads for H-Bond Assisted Cooperativity in Tandem Uncaging Systems.

"Tandem" uncaging systems, in which a photolabile protecting group (PPG) is sensitized by an energy-harvesting antenna, may increase the photosensitivity of PPGs by several orders of magnitude for two-photon (2P) photorelease. Yet, they remain poorly accessible because of arduous multi-step synthesis. In this work, we design efficient tandem uncaging systems by (i) using a convenient assembly of the building blocks relying on click chemistry, (ii) H-bonding induced proximity thus facilitating (iii) photoinduced electron transfer (PeT) as a cooperative mechanism. A strong two-photon absorber electron-donating quadrupolar antenna and various electron-accepting PPGs (mDEAC, MNI or MDNI) were clicked stepwise onto a "tweezer-shaped" pyrido-2,6-dicarboxylate platform whose H-bonding and p-stacking abilities were exploited to keep the antenna and the PPGs in close proximity. The different electron acceptor ability of the PPGs led to dyads with wildly different behaviors. Whilst the MDNI and MNI dyads showed poor dark stability or no photo-uncaging ability due to their too high electron accepting character, the mDEAC dyad benefited from optimum redox potentials to promote PeT and slow down charge recombination, resulting in enhanced uncaging quantum yield (Fu=0.38) compared to mDEAC (Fu=0.014). The unique resulted in large 2P photo-sensitivity in the near-infrared window (240 GM at 710 nm).

Ultrafast Electron-Transfer Via Hybrid States at Perovskite/Fullerene Interface.

Interfacial charge-transfer between perovskite and charge-transport layers plays a key role in determining performance of perovskite solar cells. The conventional viewpoint emphases the necessity of favorable energy-level alignment of the two components. In recent reports, efficient electron-transfer is observed from perovskite to fullerene-based electron-transport layers even when there are unfavorable energy-level alignments, but the mechanism is still unclear. Here, using an ultrafast in situ two-photon photoelectron spectroscopy, real-time observations of electron-transfer processes at CsPbI3/C60 interface in both temporal and energetic dimensions are reported. Due to strong electronic coupling, a large amount of interfacial hybrid states is generated at the interfaces, aiding fast photoinduced electron-transfer in ≈124 fs. This process is further verified by nonadiabatic molecular dynamics simulations and transient absorption experiments. The short timescale explains why electron-transfer can overcome unfavorable energy-level alignments, providing a guideline for device design.