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Despite an abundance of pharmacologic and surgical epilepsy treatments, there remain millions of patients suffering from poorly controlled seizures. One approach to closing this treatment gap may be found through a deeper mechanistic understanding of the network alterations that underly this aberrant activity. Functional optical imaging in vertebrate models provides powerful advantages to this end, enabling the spatiotemporal acquisition of individual neuron activity patterns across multiple seizures. This coupled with the advent of genetically encoded indicators, be them for specific ions, neurotransmitters or voltage, grants researchers unparalleled access to the intact nervous system. Here, we will review how in vivo functional optical imaging in various vertebrate seizure models has advanced our knowledge of seizure dynamics, principally seizure initiation, propagation and termination. [ABSTRACT FROM AUTHOR]

Calcium imaging, especially two-photon imaging, has become essential in neuroscience for studying neuronal and astrocytic activity under in vivo and in vitro conditions. Current advances in the development of calcium sensors as well as imaging hardware enable high-frequency measurements of calcium signals in hundreds of cells simultaneously. The analysis of these large datasets requires special tools and usually a certain level of programming experience. Despite advancements in calcium imaging analysis software development, significant gaps remain, particularly for data acquired at a high sampling rate that would allow for the spectral analysis of calcium signals. The FluoAnalysis MATLAB toolbox addresses these gaps by offering a comprehensive solution for analyzing simultaneously measured calcium imaging and electrophysiological data. It features both GUI-based and command-line approaches, emphasizing frequency domain analysis to reveal network-level oscillatory signals linked to single-cell activity. In addition, the toolbox puts special emphasis on differentiating between astrocytes and neurons, revealing the interactions between the network activity of the two major cell types of the brain. It facilitates a streamlined workflow for data loading, ROI identification, cell classification, fluorescence intensity calculation, spectral analysis, and report generation, supporting both manual and automated high-throughput analysis. This versatile platform enables the comprehensive analysis of large imaging datasets. In conclusion, the FluoAnalysis MATLAB toolbox provides a robust and versatile platform for the integrated analysis of calcium imaging and electrophysiological data, supporting diverse neuroscience research applications. [ABSTRACT FROM AUTHOR]

Significance: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of onephoton (1P) versus two-photon (2P) voltage imaging are not well characterized. Aim: We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach: We measure the brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in the mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results: Compared with 1P excitation, 2P excitation requires ~104-fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in the mouse cortex with a target SNR of 10 (spike height to baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of >300 µm, 2P voltage imaging using an 80-MHz source can record from no more than ~12 neurons simultaneously. Conclusions: Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at a depth of >300 µm will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging. [ABSTRACT FROM AUTHOR]

An effective, GFP‐inspired fluorescent Zn2+ sensor is developed for two–photon microscopy and related biological application that features an 8‐methoxyquinoline moiety. Excellent photophysical characteristics including a 37‐fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two–photon cross–section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn–on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc–bound compounds are presented. [ABSTRACT FROM AUTHOR]

As a unique type of supramolecular self‐assemblies, crystalline host‐guest aggregates have attracted extensive interests in multiple application fields. Herein, a crystalline host‐guest aggregate LIFM‐HG1 was obtained with curcubit[8]uril as the host and carboxypyridinium salt as the guest. Single‐crystal structural analysis indicates that the presence of abundant weak interactions in LIFM‐HG1 provides a rigid environment for the guest molecule and effectively blocks the external quenchers. Spectral analysis and theoretical calculations confirm the presence of robust triplet energy levels in LIFM‐HG1. Even more impressively, the intersystem crossing channels of the guest molecules are greatly opened up after the formation of the crystalline host‐guest aggregate, resulting in a large kisc of 6.70 × 107 s−1 at room temperature for LIFM‐HG1 (which is ∼0 for pure guest), leading to fascinating multichannel (including one‐photon, two‐photon, and X‐ray) excited LPL properties. In addition, the crystalline LIFM‐HG1 has a much higher triplet state luminescence efficiency under X‐ray and two‐photon excitation than that under single‐photon excitation (AP/AF = 86.8, 44.8, 10.7 under the three circumstances, respectively). And the phosphorescent emission intensity of LIFM‐HG1 is 27.6 times higher than that of the crystalline guest under X‐ray excitation. As a result, LIFM‐HG1 shows a long afterglow retention time under both single‐ and two‐photon excitation, and an impressive afterglow retention time of 1 s under X‐ray excitation. Furthermore, the excellent lysosomal targeting and low cytotoxicity by the formation of host‐guest aggregate makes LIFM‐HG1 promising to be used as a novel lysosomal‐targeted two‐photon excited phosphorescent tracer. [ABSTRACT FROM AUTHOR]

Significance: Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. Aim: We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. Approach: We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). Results: We describe "universal" implants for acute and chronic simultaneous brain--spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the "BLmini," which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. Conclusions: The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord--brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior. [ABSTRACT FROM AUTHOR]

The Er3+/Yb3+ co-doped Y4GeO8 crystal powders were successfully synthesized using a high-temperature solid-phase method. The crystal structure of the obtained phosphors was confirmed to be pure Y4GeO8 through X-ray diffraction (XRD) analysis. A regression equation correlating Er3+/Yb3+ doping concentrations with luminescent intensity was established based on the optimized theoretical model derived from experimental design. The optimal concentrations of Er3+ and Yb3+ under 980 nm laser excitation were determined as 7.41 and 21.34%, respectively, while under 1550 nm laser excitation, the concentrations were 2.66 and 17.42%, respectively. The fluorescence emission spectra of the up-conversion samples were measured, revealing intense green and red emissions with peaks at 542, 546, and 654 nm under 980 nm excitation, and peaks at 546, 557, and 663 nm under 1550 nm excitation. These peaks correspond to transitions from 2H11/2 to 4I15/2, 4S3/2 to 4I15/2, and 4F9/2 to 4I15/2 energy levels. The relationship between up-conversion luminescence and laser operating current for the optimal samples under 980 nm and 1550 nm was investigated, uncovering that up-conversion luminescence occurs through both two-photon and three-photon processes. A detailed analysis and discussion of the up-conversion luminescence mechanisms were conducted. Furthermore, the relationship between up-conversion fluorescence and temperature for the optimal samples was studied, revealing excellent temperature-sensing characteristics under 980 nm and 1550 nm laser excitations. The calculated illumination region coordinates for the optimal samples under 980 nm and 1550 nm wavelength excitations were (0.5558, 0.4362) and (0.5256, 0.4687), respectively. The research highlights the potential of rare-earth ion-doped up-conversion luminescent materials for diverse anti-counterfeiting applications. In particular, the Y4GeO8: Yb3+/Er3+ phosphors, incorporating a dual-excitation mechanism, enhance the security of anti-counterfeiting strategies in multifaceted scenarios. The study underscores the promising developments in this field. [ABSTRACT FROM AUTHOR]

In recent years, the development of carbon dot‐based fluorescent nanoparticles for bioimaging applications has attracted the attention of the scientific community. However, many of these systems absorb and emit in the blue–green region of the electromagnetic spectrum, limiting their application in bioimaging. Herein, the facile design and development of highly efficient two‐photon excitable red‐emissive carbon dots are reported and their high performance in bioimaging applications is demonstrated. The importance of aromatic precursors in developing red‐emissive carbon dots is demonstrated. The optimized carbon dots are highly biocompatible and nontoxic, with remarkable photostability in cells under two‐photon near‐infrared excitation. The present study points to the great potential of two‐photon excitable red‐emissive carbon dot as an efficient bioimaging agent for cellular biolabeling, long‐term and real‐time cellular imaging, and high‐resolution deep‐tissue imaging in complex biological systems. [ABSTRACT FROM AUTHOR]

Regenerative medicine, which utilizes stem cells for tissue and organ repair, holds immense promise in healthcare. A comprehensive understanding of stem cell characteristics is crucial to unlock their potential. This study explores the pivotal role of optical microscopy in advancing regenerative medicine as a potent tool for stem cell research. Advanced optical microscopy techniques enable an in‐depth examination of stem cell behavior, morphology, and functionality. The review encompasses current optical microscopy, elucidating its capabilities and constraints in stem cell imaging, while also shedding light on emerging technologies for improved stem cell visualization. Optical microscopy, complemented by techniques like fluorescence and multiphoton imaging, enhances our comprehension of stem cell dynamics. The introduction of label‐free imaging facilitates noninvasive, real‐time stem cell monitoring without external dyes or markers. By pushing the boundaries of optical microscopy, researchers reveal the intricate cellular mechanisms underpinning regenerative processes, thereby advancing more effective therapeutic strategies. The current study not only outlines the future of regenerative medicine but also underscores the pivotal role of optical microscopy in both structural and functional stem cell imaging. [ABSTRACT FROM AUTHOR]

Antibiotics are emerging contaminants in the environment, and their monitoring is important for food safety and human health. Herein, we synthesized a two-photon carbon quantum dots (TP-CQDs) nanoprobe using a simple and green hydrothermal approach and applied it as a chemosensor to detect chlortetracycline (CTC). CTC quenches the intrinsic fluorescence of TP-CQDs, providing a linear response within the 0.5 to 60 μM range and yielding a limit of detection of only 25 nM. Besides excellent stability and good optical properties, we found that TP-CQDs are selective to CTC as structurally similar antibiotics, along with cations, anions, and amino acids, had no significant influence on fluorescence emission. Finally, we successfully applied this sensing platform to quantify CTC in milk, fish, and eggs. [ABSTRACT FROM AUTHOR]

AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis. [ABSTRACT FROM AUTHOR]

Multi‐color two‐photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two‐photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two‐photon non‐linear structured illumination microscope (2P‐NLSIM), whose resolution improved three‐fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super‐resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P‐NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells. [ABSTRACT FROM AUTHOR]

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Functional nucleic acid (FNA) probes have been widely used in environmental monitoring, food analysis, clinical diagnosis, and biological imaging because of their easy synthesis, functional modification, flexible design, and stable properties. However, most FNA probes are designed based on one-photon (OP) in the ultraviolet or visible regions, and the effectiveness of these OP-based FNA probes may be hindered by certain factors, such as their potential for photodamage and limited light tissue penetration. Two-photon (TP) is characterized by the nonlinear absorption of two relatively low-energy photons of near-infrared (NIR) light with the resulting emission of high-energy ultraviolet or visible light. TP-based FNA probes have excellent properties, including lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution, making them more advantageous than the conventional OP-based FNA probes in biomedical sensing. In this review, we summarize the recent advances of TP-excited and -activated FNA probes and detail their applications in biomolecular detection. In addition, we also share our views on the highlights and limitations of TP-based FNA probes. The ultimate goal is to provide design approaches for the development of high-performance TP-based FNA probes, thereby promoting their biological applications. [ABSTRACT FROM AUTHOR]

Multiphoton absorption of entangled photons offers ways for obtaining unique information about chemical and biological processes. Measurements with entangled photons may enable sensing biological signatures with high selectivity and at very low light levels to protect against photodamage. In this paper, we present a theoretical and experimental study of the excitation wavelength dependence of the entangled two-photon absorption (ETPA) process in a molecular system, which provides insights into how entanglement affects molecular spectra. We demonstrate that the ETPA excitation spectrum can be different from that of classical TPA as well as that for one-photon resonant absorption (OPA) with photons of doubled frequency. These results are modeled by assuming the ETPA cross-section is governed by a two-photon excited state radiative linewidth rather than by electron-phonon interactions, and this leads to excitation spectra that match the observed results. Further, we find that the two-photon-allowed states with highest TPA and ETPA intensities have high electronic entanglements, with ETPA especially favoring states with the longest radiative lifetimes. These results provide concepts for the development of quantum light--based spectroscopy and microscopy that will lead to much higher efficiency of ETPA sensors and low- intensity detection schemes. [ABSTRACT FROM AUTHOR]

To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon trackingmicroscope recorded fromindividual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D randomaccess scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning. [ABSTRACT FROM AUTHOR]

Different segments of the cerebral vascular network may react distinctly to brain ischemia and recanalization. However, there are limited systematic observations of these vascular responses in mice under a physiological state following ischemic stroke. Herein, we aimed to investigate the vasodynamics among several segments along the cerebral vessels in awake mice following cerebral ischemia/recanalization via two-photon imaging. Plasma in the blood vessels were labelled with fluorescein isothiocyanate dextran. Smooth muscle cells and pericytes were labelled via a genetic mouse line (PDGFRβ-tdTomato). We observed a no-reflow phenomenon in downstream microcirculation, and the vasodynamics of different segments of larger cerebral vessels varied in the penumbra area following cerebral ischemia-reperfusion. Despite obtaining reperfusion from the middle cerebral artery, there were significant constrictions of the downstream blood vessels in the ischemic penumbra zone. Interestingly, we observed an extensive constriction of the capillaries 3 hours following recanalization, both at the site covered by pericyte soma and by the pericyte process alone. In addition, we did not observe a significant positive correlation between the changed capillary diameter and pericyte coverage along the capillary. Taken together, abnormal constrictions and vasodynamics of cerebral large and small vessels may directly contribute to microcirculation failure following recanalization in ischemic stroke. [ABSTRACT FROM AUTHOR]

Cytochrome P450 1A is one of the vital subfamilies of heme-containing cytochrome P450 enzymes belonging to an important exogenous metabolizing CYP in human. The abnormal of endoplasmic reticulum (ER) may directly affect the functional activity of ER-located CYP1A and be associated with the occurrence and development of various diseases. In the present study, we constructed a selective two-photon fluorescent probe ERNM for rapid and visual detection of endogenous CYP1A that was localized in the ER. ERNM could target the ER and detect the enzymatically active CYP1A in living cells and tissues. The monitoring ability of ERNM for the fluctuations in functionality level of CYP1A was confirmed using ER stressed A549 cell. Based on the ER-targeting two-photon probe for CYP1A, the close association of ER state and the functional activity of ER-locating CYP1A was confirmed, which would promote the deep understanding of the biofunction of CYP1A in various ER-related diseases. [ABSTRACT FROM AUTHOR]

Our understanding of molecules has stagnated at a single quantum system, with atoms as Newtonian particles and electrons as quantum particles. Here, however, we reveal that both atoms and electrons in a molecule are quantum particles, and their quantum–quantum interactions create a previously unknown, newfangled molecular property—supracence. Molecular supracence is a phenomenon in which the molecule transfers its potential energy from quantum atoms to photo-excited electrons so that the emitted photon has more energy than that of the absorbed one. Importantly, experiments reveal such quantum energy exchanges are independent of temperature. When quantum fluctuation results in absorbing low-energy photons, yet still emitting high-energy photons, supracence occurs. This report, therefore, reveals novel principles governing molecular supracence via experiments that were rationalized by full quantum (FQ) theory. This advancement in understanding predicts the super-spectral resolution of supracence, and molecular imaging confirms such innovative forecasts using closely emitting rhodamine 123 and rhodamine B in living cell imaging of mitochondria and endosomes. [ABSTRACT FROM AUTHOR]

In recent years, the development of novel photocrosslinking strategies and photoactivatable materials has stimulated widespread use of light-mediated biofabrication techniques. However, despite great progress toward more efficient and biocompatible photochemical strategies, current photoresins still rely on photoinitiators (PIs) producing radical-initiating species to trigger the so-called free-radical crosslinking/polymerization. In the context of bioprinting, where cells are encapsulated in the bioink, the presence of radicals raises concerns of potential cytotoxicity. In this work, a universal, radical-free (RF) photocrosslinking strategy to be used for light-based technologies is presented. Leveraging RF uncaging mechanisms and Michael addition, cell-laden constructs are photocrosslinked by means of one- and two-photon excitation with high biocompatibility. A hydrophilic coumarin-based group is used to cage a universal RF photocrosslinker based on 4-arm-PEG-thiol (PEG4SH). Upon light exposure, thiols are uncaged and react with an alkene counterpart to form a hydrogel. RF photocrosslinker is shown to be highly stable, enabling potential for off-the-shelf products. While PI-based systems cause a strong upregulation of reactive oxygen species (ROS)-associated genes, ROS are not detected in RF photoresins. Finally, optimized RF photoresin is successfully exploited for high resolution two-photon stereolithography (2P-SL) using remarkably low polymer concentration (<1.5%), paving the way for a shift toward radical-free light-based bioprinting. [ABSTRACT FROM AUTHOR]

In order to obtain the maximum green up-conversion luminescence intensity of Ho3+/Yb3+ co-doped YNbO4, a uniform design and a quadratic general rotation combination design were applied to optimize the doping concentration. The concentration of the rare earth corresponding to the strongest green luminescence intensity was determined to be 10% Ho3+/34.9% Yb3+. Maximum luminous intensity of green light tested under 980 nm excitation was 157373.266, which was close to the theoretical calculated integral intensity value of 157290.825. The variation of the up-conversion luminescence spectra at different power density of the 980 nm laser was characterized. According to the formula fitting, the up-conversion luminescence process is a two-photon process. Meanwhile, the temperature sensing characteristic of the samples has been discussed. Finally, the CIE coordinates were analyzed and calculated to be (0.297, 0.692). [ABSTRACT FROM AUTHOR]

Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies. Nonetheless, the field of CAR T-cells is currently facing several major challenges. In particular, the CAR T-cell strategy has not yet produced favorable clinical responses when targeting solid tumors. In this context, it is of paramount importance to understand the determinants that limit the efficacy of T-cell-based immunotherapy. Characterization of CAR T-cells is usually based on flow cytometry and whole-transcriptome profiling. These approaches have been very valuable to determine intrinsic elements that condition T-cell ability to proliferate and expand. However, they do not take into account spatial and kinetic aspects of T-cell responses. In particular, in order to control tumor growth, CAR T-cells need to enter into the tumor, migrate within a complex tumor environment, and form productive conjugates with their targets. Advanced imaging techniques combined with innovative preclinical models represent promising tools to uncover the dynamics of CAR T-cells. In this review, we will discuss recent results on the biology of engineered T-cells that have been obtained with real-time imaging microscopy. Important notions have emerged from these imaging-based studies, such as the multi-killing potential of CAR T-cells. Finally, we will highlight how imaging techniques combined with other tools can solve remaining unresolved questions in the field of engineered T-cells. [ABSTRACT FROM AUTHOR]

Sensory cortices, even of primary regions, are not purely unisensory. Rather, cortical neurons in sensory cortex show various forms of multisensory interactions. While some multisensory interactions naturally co-occur, the combination of others will co-occur through experience. In real life, learning and experience will result in conjunction with seemingly disparate sensory information that ultimately becomes behaviorally relevant, impacting perception, cognition, and action. Here we describe a novel auditory discrimination task in mice, designed to manipulate the expectation of upcoming trials using olfactory cues. We show that, after learning, female mice display a transient period of several days during which they exploit odor-mediated expectations for making correct decisions. Using two-photon calcium imaging of single neurons in auditory cortex (ACx) during behavior, we found that the behavioral effects of odor-mediated expectations are accompanied by an odor-induced modulation of neuronal activity. Further, we find that these effects are manifested differentially, based on the response preference of individual cells. A significant portion of effects, but not all, are consistent with a predictive coding framework. Our data show that learning novel odor-sound associations evoke changes in ACx. We suggest that behaviorally relevant multisensory environments mediate contextual effects as early as ACx. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) is thought to play a role in driving the rapid, spontaneous brain-state transitions that occur during wakefulness; however, the spatiotemporal properties of cortical ACh activity during these state changes are still unclear. We perform simultaneous imaging of GRAB-ACh sensors, GCaMP-expressing basal forebrain axons, and behavior to address this question. We observed a high correlation between axon and GRAB-ACh activity around periods of locomotion and pupil dilation. GRAB-ACh fluorescence could be accurately predicted from axonal activity alone, and local ACh activity decreased at farther distances from an axon. Deconvolution of GRAB-ACh traces allowed us to account for sensor kinetics and emphasized rapid clearance of small ACh transients. We trained a model to predict ACh from pupil size and running speed, which generalized well to unseen data. These results contribute to a growing understanding of the precise timing and spatial characteristics of cortical ACh during fast brain-state transitions. [Display omitted] • Bulk ACh release around cortical cholinergic axons can be observed in vivo • ACh levels scale with locomotion speed and pupil size • Sensor deconvolution allows evaluation of precise ACh temporal dynamics • ACh activity can be predicted from axon activity and behavioral variables Neyhart et al. describe precise timing and spatial characteristics of cortical acetylcholine availability in relation to cholinergic axon activity and behavioral markers. [ABSTRACT FROM AUTHOR]

Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 lm. It is at this scale where most of the unknown and exciting action is and where cell biologists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its performance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy techniques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia. [ABSTRACT FROM AUTHOR]

Carbon dots (CDs) have gradually become a widely favored type of fluorescent nanomaterials with unlimited and promising applications. This work reports a means of breaking the shackle of method to develop new red and white CDs by introducing free radicals. The resulting white emissive CDs in this strategy are employed to demonstrate electroluminescent white-light-emitting diodes (WLEDs) and achieve a record-high external quantum efficiency (0.95%) of one-step-produced white CDs (WCD)-LEDs, which dramatically simplifies the whole fabrication processes of WLEDs. With additional passivation treatment, the red CDs (RCD2) with excellent properties such as N and S co-doping, bright (quantum yield = 49%) and stable photoluminescence (PL), large positive zeta potential (+20.5 mV), and two-photon fluorescence are obtained. Such RCD2 are used for rapid staining (5 min) of yeast cells. The two CDs synthesized via this method have outstanding performance in different aspects, which provides new promise of CDs for further functionalization and applications. [ABSTRACT FROM AUTHOR]

A series of dinuclear RuII complexes with extremely high TPA cross sections in the range of 800–900 nm have been designed. The amphiphilic complex Ru3 containing tert‐butyl groups has balanced performance in singlet oxygen generation and photothermal conversion and becomes the ideal drug candidate of the series. Ru3 targets mitochondria without penetrating the nucleus, which substantially increases its photodynamic therapy activity and reduces its dark cytotoxicity. Ru3 successfully suppresses melanoma tumor growth in vitro and in vivo with combined photodynamic and photothermal therapy under low light dose irradiation of an 808 nm low‐power laser, avoiding the known PDT resistance in melanoma. The excellent therapeutic effect of Ru3 facilitates its applications in further human trials for larger or deeper buried tumors, thereby becoming a prospective candidate for a new generation of low‐power IR‐driven dual PDT/PTT drugs. [ABSTRACT FROM AUTHOR]

A series of dinuclear RuII complexes with extremely high TPA cross sections in the range of 800–900 nm have been designed. The amphiphilic complex Ru3 containing tert‐butyl groups has balanced performance in singlet oxygen generation and photothermal conversion and becomes the ideal drug candidate of the series. Ru3 targets mitochondria without penetrating the nucleus, which substantially increases its photodynamic therapy activity and reduces its dark cytotoxicity. Ru3 successfully suppresses melanoma tumor growth in vitro and in vivo with combined photodynamic and photothermal therapy under low light dose irradiation of an 808 nm low‐power laser, avoiding the known PDT resistance in melanoma. The excellent therapeutic effect of Ru3 facilitates its applications in further human trials for larger or deeper buried tumors, thereby becoming a prospective candidate for a new generation of low‐power IR‐driven dual PDT/PTT drugs. [ABSTRACT FROM AUTHOR]

Neuronal hippocampal Ca2+ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer's disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca2+ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca2+ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single‐cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging‐ and Ca2+‐dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L‐type voltage‐gated Ca2+ channel modifier Bay‐K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca2+ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age. [ABSTRACT FROM AUTHOR]

Treatment of brain disorders relies on efficient delivery of therapeutics to the brain, which is hindered by the blood–brain barrier (BBB). The work of Prof. Margareta Hammarlund-Udenaes was instrumental in understanding the principles of drug delivery to the brain and developing new tools to study it. Here, we show how some of the concepts developed in her research can be translated to in vivo 2-photon microscopy (2PM) studies of the BBB. We primarily focus on the methods developed in our laboratory to characterize the paracellular diffusion, adsorptive-mediated transcytosis, and receptor-mediated transcytosis of drug nanocarriers at the microscale, illustrating how 2PM can deepen our understanding of the mechanisms of drug delivery to the brain. [ABSTRACT FROM AUTHOR]

Basal dendrites of layer 5 cortical pyramidal neurons exhibit Na+ and N -methyl-D-aspartate receptor (NMDAR) regenerative spikes and are uniquely poised to influence somatic output. Nevertheless, due to technical limitations, how multibranch basal dendritic integration shapes and enables multiplexed barcoding of synaptic streams remains poorly mapped. Here, we combine 3D two-photon holographic transmitter uncaging, whole-cell dynamic clamp, and biophysical modeling to reveal how synchronously activated synapses (distributed and clustered) across multiple basal dendritic branches are multiplexed under quiescent and in vivo -like conditions. While dendritic regenerative Na+ spikes promote millisecond somatic spike precision, distributed synaptic inputs and NMDAR spikes regulate gain. These concomitantly occurring dendritic nonlinearities enable multiplexed information transfer amid an ongoing noisy background, including under back-propagating voltage resets, by barcoding the axo-somatic spike structure. Our results unveil a multibranch dendritic integration framework in which dendritic nonlinearities are critical for multiplexing different spatial-temporal synaptic input patterns, enabling optimal feature binding. [Display omitted] • 3D two-photon holographic uncaging assays multibranch basal dendritic integration • Dendritic nonlinearities facilitate barcoding via spike timing and rate modulation • Barcoding enables multibranch multiplexing under quiescent and in vivo -like states • Dendritic Na+ spikes are critical for pattern classification Xiao et al. use 3D two-photon holography, dynamic clamp, pharmacology, and modeling to unravel multibranch dendritic integration in layer 5 pyramidal neurons under quiescent and in vivo -like states. They reveal a critical role of dendritic nonlinearities in barcoding the somatic spike structure, allowing for optimal feature binding and pattern classification. [ABSTRACT FROM AUTHOR]

Lysyl oxidase (LOX) is a copper-binding enzyme that cross-links elastin and collagen. The dominant LOX variation contributes to familial thoracic aortic aneurysm. Previously reported murine Lox mutants had a mild phenotype and did not dilate without drug-induced provocation. Here, we present a new, more severe mutant, Loxb2b370.2Clo (c.G854T; p.Cys285Phe), whose mutation falls just N-terminal to the copper-binding domain. Unlike the other mutants, the C285F Lox protein was stably produced/secreted, and male C57Bl/6J Lox+/C285F mice exhibit increased systolic blood pressure (BP; p < 0.05) and reduced caliber aortas (p < 0.01 at 100mmHg) at 3 months that independently dilate by 6 months (p < 0.0001). Multimodal imaging reveals markedly irregular elastic sheets in the mutant (p = 2.8 × 10−8 for breaks by histology) that become increasingly disrupted with age (p < 0.05) and breeding into a high BP background (p = 6.8 × 10−4). Aortic dilation was amplified in males vs. females (p < 0.0001 at 100mmHg) and ameliorated by castration. The transcriptome of young Lox mutants showed alteration in dexamethasone (p = 9.83 × 10−30) and TGFβ-responsive genes (p = 7.42 × 10−29), and aortas from older C57Bl/6J Lox+/C285F mice showed both enhanced susceptibility to elastase (p < 0.01 by ANOVA) and increased deposition of aggrecan (p < 0.05). These findings suggest that the secreted Lox+/C285F mutants produce dysfunctional elastic fibers that show increased susceptibility to proteolytic damage. Over time, the progressive weakening of the connective tissue, modified by sex and blood pressure, leads to worsening aortic disease. [ABSTRACT FROM AUTHOR]

Two‐photon interference lithography with an elastomeric phase grating that allows spontaneous conformal contact to the photoresist can be used to fabricate practical‐scale, 3D photonic crystals through a single exposure. In principle, the unit cell symmetry and fill factor of the 3D periodic nanostructures produced using this technique are strongly depend on the structural parameters of the phase grating. However, theoretical approaches to derive the optimal parameters of the phase grating for realizing high‐definition 3D nanostructures are still lacking. Here, the change in the Talbot interference pattern is systematically predicted with the relief height of the phase grating under the conditions in which two‐photon interference lithography is performed through optical simulations. The collective set of results reveals the design error of the relief height of the phase grating, as inferred by the scalar approximation thus far, and presents the optimal condition that satisfies the π‐phase shift inferred by the full‐vectorial numerical solution. [ABSTRACT FROM AUTHOR]

As an important reactive oxygen species (ROS), hydrogen peroxide plays a significant role in the life activity system, and its abnormal levels are closely related to many diseases. Developing effective fluorescent probes for detecting hydrogen peroxide is very urgent. Therefore, we constructed a probe Z that can detect hydrogen peroxide in ratio. It has naphthimide as the fluorophore and phenylboronic acid pinacol esters as the recognition group. It shows higher sensitivity, lower detection limit, higher selectivity, and broad pH applicability. Moreover, probe Z has low cytotoxicity that can be used to detect exogenous hydrogen peroxide in HeLa cells and might be a potential tool for studying hydrogen peroxide in physiological activities. [ABSTRACT FROM AUTHOR]

Designing new oxygenation nanomaterials by oxygen-generating or oxygen-carrying strategies in hypoxia-associated anti-tumor therapy is a high priority target yet challenge. In this work, we fabricated a nanoplatform involving Fenton-like reaction, Pd@MOF-525@HA, to relieve tumor hypoxia via oxygen-generating strategy for enhanced oxygen-dependent anti-tumor therapy. Thereinto, the porphyrinic MOF-525 can produce singlet oxygen (1O2) via light or ultrasonic irradiation for photodynamic and sonodynamic therapy. Notably, the well-dispersed Pd nanocubes within MOF-525 can convert H2O2 into O2 to mitigate the hypoxic environment for enhanced therapy outcome. Moreover, the two-photon activity and cancer cell specific targeting capability of Pd@MOF-525@HA gave rise to deeper tissue penetration and near-infrared light-induced fluorescence imaging to achieve precise guidance for cancer therapy. This work provides a feasible way in designing new oxygenation nanomaterials to relieve tumor hypoxia for enhanced cancer treatment. [ABSTRACT FROM AUTHOR]

Epilepsy accounts for a significant proportion of the world's disease burden. Indeed, many research efforts are produced both to investigate the basic mechanism ruling its genesis and to find more effective therapies. In this framework, the use of zebrafish larvae, owing to their peculiar features, offers a great opportunity. Here, we employ transgenic zebrafish larvae expressing GCaMP6s in all neurons to characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using a custom two-photon light-sheet microscope, we perform fast volumetric functional imaging of the entire larval brain, investigating how different brain regions contribute to seizure onset and propagation. Moreover, employing a custom behavioral tracking system, we outline the progressive alteration of larval swim kinematics, resulting from different grades of seizures. Collectively, our results show that the epileptic larval brain undergoes transitions between diverse neuronal activity regimes. Moreover, we observe that different brain regions are progressively recruited into the generation of seizures of diverse severity. We demonstrate that midbrain regions exhibit highest susceptibility to the convulsant effects and that, during periods preceding abrupt hypersynchronous paroxysmal activity, they show a consistent increase in functional connectivity. These aspects, coupled with the hub-like role that these regions exert, represent important cues in their identification as epileptogenic hubs. [ABSTRACT FROM AUTHOR]

Simple Summary: Photodynamic therapy (PDT) is a light-activated treatment modality, which is being clinically used and further developed for a number of premalignancies, solid tumors, and disseminated cancers. Nanomedicines that facilitate PDT (photonanomedicines, PNMs) have transformed its safety, efficacy, and capacity for multifunctionality. This review focuses on the state of the art in deep-tissue activation technologies for PNMs and explores how their preclinical use can evolve towards clinical translation by harnessing current clinically available instrumentation. With the continued development of nanomaterials over the past two decades, specialized photonanomedicines (light-activable nanomedicines, PNMs) have evolved to become excitable by alternative energy sources that typically penetrate tissue deeper than visible light. These sources include electromagnetic radiation lying outside the visible near-infrared spectrum, high energy particles, and acoustic waves, amongst others. Various direct activation mechanisms have leveraged unique facets of specialized nanomaterials, such as upconversion, scintillation, and radiosensitization, as well as several others, in order to activate PNMs. Other indirect activation mechanisms have leveraged the effect of the interaction of deeply penetrating energy sources with tissue in order to activate proximal PNMs. These indirect mechanisms include sonoluminescence and Cerenkov radiation. Such direct and indirect deep-tissue activation has been explored extensively in the preclinical setting to facilitate deep-tissue anticancer photodynamic therapy (PDT); however, clinical translation of these approaches is yet to be explored. This review provides a summary of the state of the art in deep-tissue excitation of PNMs and explores the translatability of such excitation mechanisms towards their clinical adoption. A special emphasis is placed on how current clinical instrumentation can be repurposed to achieve deep-tissue PDT with the mechanisms discussed in this review, thereby further expediting the translation of these highly promising strategies. [ABSTRACT FROM AUTHOR]

Graph: Figure 3: Polarization-dependent PL properties of Au@oligomer hybrid NPs.(a) UV-vis extinction spectra of Au@oligomer/MB, Au@oligomer, and Au NPs. As plasmon-enhanced PL is highly related to the alignment of the electric field direction and molecular dipole, polarization-directed anisotropic etching also controls the spatial distribution of the dye molecules in an asymmetric manner, which results in polarization-dependent excitation of single plasmonic hybrid NP. However, the MB molecules in the perpendicular direction of the polarization remain almost intact, which show a prominent PL if excitation polarization is aligned along the long axis of Au@oligomer hybrid NP (Figure 3(e)). Keywords: nanoparticles; nanorods; plasmons; polarization; two-photon; photolithography EN nanoparticles nanorods plasmons polarization two-photon photolithography 1003 1009 7 03/11/22 20220201 NES 220201 1 Introduction Anisotropic nanoparticles (NPs) have attracted great research interests due to their pivotal role in material performances, which can potentially lead to compelling electric, photonic, mechanical, and biological functionalities [[1]]. [Extracted from the article]

The superior colliculus (SC) is the midbrain center for integrating visual and multimodal sensory information. Neurons in the SC exhibit direction and orientation selectivity. Recent studies reported that neurons with similar preferences formed clusters in the mouse SC (Ahmadlou and Heimel, 2015; Feinberg and Meister, 2015; de Malmazet et al., 2018; Li et al., 2020). However, it remains controversial as to how these clusters are organized within the SC (Inayat et al., 2015; Chen et al., 2021). Here, we found that different brain states (i.e., awake or anesthetized with isoflurane) changed the selectivity of individual SC neurons and organizations of the neuronal population in both male and female mice. Using two-photon Ca2+ imaging, we examined both individual neuronal responses and the spatial patterns of their population responses. Under isoflurane anesthesia, orientation selectivity increased and a larger number of orientation-selective cells were observed when compared with the awake condition, whereas the proportions of direction-selective cells were similar in both conditions. Furthermore, direction- and orientation-selective cells located at closer positions showed more similar preferences, and cluster- like spatial patterns were enhanced. Inhibitory responses of direction-selective neurons were also reduced under isoflurane anesthesia. Thus, the changes in the spatial organization of response patterns were considered to be because of changes in the balance of excitation and inhibition, with excitation dominance, in the local circuits. These results provide new insights into the possibility that the functional organization of feature selectivity in the brain is affected by brain state. [ABSTRACT FROM AUTHOR]

Wireless and precise stimulation of deep brain structures could have important applications to study intact brain circuits and treat neurological disorders. Herein, we report that magnetoelectric nanoparticles (MENs) can be guided to a targeted brain region to stimulate brain activity with a magnetic field. We demonstrated the nanoparticles' capability to reliably evoke fast neuronal responses in cortical slices ex vivo. After fluorescently labeled MENs were intravenously injected and delivered to a targeted brain region by applying a magnetic field gradient, a magnetic field of low intensity (350–450 Oe) applied to the mouse head reliably evoked cortical activities, as revealed by two-photon and mesoscopic imaging of calcium signals and by an increased number of c-Fos expressing cells after stimulation. Neither brain delivery of MENs nor the magnetic stimulation caused significant increases in astrocytes and microglia. Thus, MENs could enable a non-invasive and contactless deep brain stimulation without the need of genetic manipulation. [ABSTRACT FROM AUTHOR]

Identification of neurons undergoing plasticity in response to external stimuli is one of the pertinent problems in neuroscience. Immediate early genes (IEGs) are widely used as a marker for neuronal plasticity. Here, we model the dynamics of IEG expression as a consecutive, irreversible first-order reaction with a limiting substrate. First, we develop an analytical framework to show that such a model, together with two-photon in vivo imaging of IEG expression, can be used to identify distinct neuronal subsets representing multiple memories. Using the above combination, we show that the expression kinetics, rather than intensity threshold, can be used to identify neuronal ensembles responding to the presentation of two events in vivo. The analytical expression allowed us to segregate the neurons based on their temporal response to one specific behavioural event, thereby improving the ability to detect plasticity related neurons. We image the retrosplenial cortex (RSc) of cfos-GFP transgenic mice to follow the dynamics of cellular changes resulting from contextual fear conditioning behaviour, enabling us to establish a representation of context in RSc at the cellular scale following memory acquisition. Thus, we obtain a general method that distinguishes neurons that took part in multiple temporally separated events by measuring fluorescence of individual neurons in live mice. [ABSTRACT FROM AUTHOR]

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies. [ABSTRACT FROM AUTHOR]

The novel theranostic nanosystems based on two‐photon fluorescence can achieve higher spatial resolution of deep tissue imaging for simultaneous diagnosis and therapy of a variety of cancers. Herein, we have designed and prepared FRET‐based two‐photon mesoporous silica nanoparticles (MTP–MSNs) for single‐excitation multiplexed intracellular imaging and targeted cancer therapy for the first time. This nanosystem includes two constituents, containing (1) multicolor two‐photon mesoporous silica nanoparticles and (2) cancer cell‐targeting aptamers that act as gatekeepers for MTP–MSNs. After incubation with cancer cells, the Dox‐loaded and aptamer‐capped MTP–MSNs could be internalized into the cells, opening the pores and releasing the drug. Furthermore, using two‐photon multicolor fluorescence, MTP–MSNs could serve as good contrast agents for multicolor two‐photon intracellular imaging with increased imaging depth and improved spatial localization of tissue. In sum, these multicolor MTP–MSNs provide a promising system for traceable targeted cancer therapy with further applications in multiplex intracellular imaging and the screening of drug. [ABSTRACT FROM AUTHOR]

The novel theranostic nanosystems based on two‐photon fluorescence can achieve higher spatial resolution of deep tissue imaging for simultaneous diagnosis and therapy of a variety of cancers. Herein, we have designed and prepared FRET‐based two‐photon mesoporous silica nanoparticles (MTP–MSNs) for single‐excitation multiplexed intracellular imaging and targeted cancer therapy for the first time. This nanosystem includes two constituents, containing (1) multicolor two‐photon mesoporous silica nanoparticles and (2) cancer cell‐targeting aptamers that act as gatekeepers for MTP–MSNs. After incubation with cancer cells, the Dox‐loaded and aptamer‐capped MTP–MSNs could be internalized into the cells, opening the pores and releasing the drug. Furthermore, using two‐photon multicolor fluorescence, MTP–MSNs could serve as good contrast agents for multicolor two‐photon intracellular imaging with increased imaging depth and improved spatial localization of tissue. In sum, these multicolor MTP–MSNs provide a promising system for traceable targeted cancer therapy with further applications in multiplex intracellular imaging and the screening of drug. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function. [ABSTRACT FROM AUTHOR]

Despite extensive research, the exact pathomechanisms associated with epileptic seizure formation and propagation have not been elucidated completely. Two-photon imaging (2PI) is a fluorescence-based microscopy technique that, over the years, has been used to evaluate pathomechanisms associated with epileptic seizures and epilepsy. Here, we review previous applications of 2PI in epilepsy. A systematic search was performed in multiple literature databases. We identified 38 publications that applied 2PI in epilepsy research. These studies described models of epileptic seizure propagation; anatomical changes and functional alterations of microglia, astrocytes, and neurites; and neurometabolic effects that accompany seizures. Moreover, various neurovascular alterations that accompany seizure onset and ictal events, such as blood vessel responses, have been visualized using 2PI. Lastly, imaging and quantitative analysis of oxidative stress and the aggregation of lipofuscin in the neurovasculature have been accomplished with 2PI. Cumulatively, these papers and their reported findings demonstrate that 2PI is an especially well-suited imaging technique in the domain of epilepsy research, and these studies have significantly improved our understanding of the disorder. The application of 2PI provides ample possibilities for future research, most interestingly on human brains, while also stretching beyond the field of epilepsy. [ABSTRACT FROM AUTHOR]

The hippocampal place cell system in rodents has provided a major paradigm for the scientific investigation of memory function and dysfunction. Place cells have been observed in area CA1 of the hippocampus of both freely moving animals, and of head-fixed animals navigating in virtual reality environments. However, spatial coding in virtual reality preparations has been observed to be impaired. Here we show that the use of a real-world environment system for head-fixed mice, consisting of an air-floating track with proximal cues, provides some advantages over virtual reality systems for the study of spatial memory. We imaged the hippocampus of head-fixed mice injected with the genetically encoded calcium indicator GCaMP6s while they navigated circularly constrained or open environments on the floating platform. We observed consistent place tuning in a substantial fraction of cells despite the absence of distal visual cues. Place fields remapped when animals entered a different environment. When animals re-entered the same environment, place fields typically remapped over a time period of multiple days, faster than in freely moving preparations, but comparable with virtual reality. Spatial information rates were within the range observed in freely moving mice. Manifold analysis indicated that spatial information could be extracted from a low-dimensional subspace of the neural population dynamics. This is the first demonstration of place cells in head-fixed mice navigating on an air-lifted real-world platform, validating its use for the study of brain circuits involved in memory and affected by neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

There has been considerable research showing populations of neurons encoding for different aspects of space in the brain. Recently, several studies using two-photon calcium imaging and virtual navigation have identified "spatially" modulated neurons in the posterior cortex. We enquire here whether the presence of such spatial representations may be a cortex-wide phenomenon and, if so, whether these representations can be organized in the absence of the hippocampus. To this end, we imaged the dorsal cortex of mice running on a treadmill populated with tactile cues. A high percentage (40-80%) of the detected neurons exhibited sparse, spatially localized activity, with activity fields uniformly localized over the track. The development of this location specificity was impaired by hippocampal damage. Thus, there is a substantial population of neurons distributed widely over the cortex that collectively form a continuous representation of the explored environment, and hippocampal outflow is necessary to organize this phenomenon. [ABSTRACT FROM AUTHOR]

Synapse loss is the strongest correlate for cognitive decline in Alzheimer's disease. The mechanisms underlying synapse loss have been extensively investigated using mouse models expressing genes with human familial Alzheimer's disease mutations. In this review, we summarize how multiphoton in vivo imaging has improved our understanding of synapse loss mechanisms associated with excessive amyloid in the living animal brain. We also discuss evidence obtained from these imaging studies for the role of cell-intrinsic calcium dyshomeostasis and cell-extrinsic activities of microglia, which are the immune cells of the brain, in mediating synapse loss. [ABSTRACT FROM AUTHOR]