Two-photon live imaging of direct glia-to-neuron conversion in the mouse cortex

Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for n euro regeneration in the adult mammalian central ne rvous system.Howeve r,many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry.In addition,concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tra cing mice.In this study,we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ecto pic expression of the neural transcription factor NeuroD1 in both prolife rating reactive astrocytes and lineage-tra ced astrocytes in the mouse cortex.Time-lapse imaging over several wee ks revealed the ste p-by-step transition from a typical astrocyte with numero us short,tapered branches to a typical neuro n with a few long neurites and dynamic growth cones that actively explored the local environment.In addition,these lineage-converting cells were able to migrate ra dially or to ngentially to relocate to suitable positions.Furthermore,two-photon Ca2+imaging and patch-clamp recordings confirmed that the newly generated neuro ns exhibited synchronous calcium signals,repetitive action potentials,and spontaneous synaptic responses,suggesting that they had made functional synaptic connections within local neural circuits.In conclusion,we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuro regeneration and neural circuit reconstruction.

Two-photon polymerization lithography for imaging optics

Optical imaging systems have greatly extended human visual capabilities,enabling the observation and understanding of diverse phenomena.Imaging technologies span a broad spectrum of wavelengths from x-ray to radio frequencies and impact research activities and our daily lives.Traditional glass lenses are fabricated through a series of complex processes,while polymers offer versatility and ease of production.However,modern applications often require complex lens assemblies,driving the need for miniaturization and advanced designs with micro-and nanoscale features to surpass the capabilities of traditional fabrication methods.Three-dimensional(3D)printing,or additive manufacturing,presents a solution to these challenges with benefits of rapid prototyping,customized geometries,and efficient production,particularly suited for miniaturized optical imaging devices.Various 3D printing methods have demonstrated advantages over traditional counterparts,yet challenges remain in achieving nanoscale resolutions.Two-photon polymerization lithography(TPL),a nanoscale 3D printing technique,enables the fabrication of intricate structures beyond the optical diffraction limit via the nonlinear process of two-photon absorption within liquid resin.It offers unprecedented abilities,e.g.alignment-free fabrication,micro-and nanoscale capabilities,and rapid prototyping of almost arbitrary complex 3D nanostructures.In this review,we emphasize the importance of the criteria for optical performance evaluation of imaging devices,discuss material properties relevant to TPL,fabrication techniques,and highlight the application of TPL in optical imaging.As the first panoramic review on this topic,it will equip researchers with foundational knowledge and recent advancements of TPL for imaging optics,promoting a deeper understanding of the field.By leveraging on its high-resolution capability,extensive material range,and true 3D processing,alongside advances in materials,fabrication,and design,we envisage disruptive solutions to current challenges and a promising incorporation of TPL in future optical imaging applications.

In vivo imaging of the neuronal response to spinal cord injury:a narrative review

Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron imaging.

In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons

Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain.Two-photon Ca^(2+)imaging in vivo using a cranial window and specific neuronal labeling enables realtime,in situ,and long-term imaging of the living brain.Here,we constructed a recombinant rabies virus containing the Ca^(2+)indicator GCaMP6 s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6 s signal reliability.This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca^(2+)activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca^(2+)imaging.Notably,we were able to record single-spine spontaneous Ca2+activity in specific circuits.Distinct spontaneous Ca^(2+)dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits.Our method can be applied to monitor Ca^(2+)dynamics in specific input circuits in vivo,and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

A visualization pipeline for in vivo two-photon volumetric astrocytic calcium imaging

Astrocytes,the multi-functional glial cells with the most abundant population in the brain,integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities.Astrocytic calcium(Ca^(2+))signaling is the major readout of cellular functional state of astrocytes.The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca^(2+)signals,which leaves>80%spatial information undetected.To fully probe the Ca^(2+)activity across the whole astrocytic territory,we developed a pipeline for imaging and visualizing volumetric astrocytic Ca^(2+)time-lapse images.With the pipeline,we discovered a new signal distribution pattern from three-dimensional(3D)astrocytic Ca^(2+)imaging data of mice under isoflurane anesthetic states.The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.

Femtosecond Two-Photon Detachment of Cu^- Studied By Photoelectron Imaging

The wavelength dependence of photoelectron angular distributions （PADs） of two-photon detachment of Cu^- has been directly studied by using the photoelectron map imaging. Results show that for the laser field intensity of 6.0×10^10W/cm^2, PADs exhibit dramatic change with the external field wavelength. Comparison between the experimental observation and the lowest-order perturbation theory prediction indicates that the pattern of PADs can be explained by the interference of the s and d partial waves in the final state. Relative contri- butions of s and d partial waves in the two-photon detachment at different laser wavelengths are obtained.

Facile Approach to Synthesize Gold Nanorod@Polyacrylic Acid/Calcium Phosphate Yolk–Shell Nanoparticles for Dual-Mode Imaging and pH/NIR-Responsive Drug Delivery

A facile strategy to fabricate gold nanorod@polyacrylic acid/calcium phosphate(Au NR@-PAA/Ca P) yolk–shell nanoparticles(NPs) composed with a PAA/Ca P shell and an Au NR yolk is reported. The asobtained Au NR@PAA/Ca P yolk–shell NPs possess ultrahigh doxorubicin(DOX) loading capability(1 mg DOX/mg NPs), superior photothermal conversion property(26%)and p H/near-infrared(NIR) dual-responsive drug delivery performance. The released DOX continuously increased due to the damage of the Ca P shell at low p H values. When the DOX-loaded Au NR@PAA/Ca P yolk–shell NPs wereexposed to NIR irradiation, a burst-like drug release occurs owing to the heat produced by the Au NRs. Furthermore,Au NR@PAA/Ca P yolk–shell NPs are successfully employed for synergic dual-mode X-ray computed tomography/photoacoustic imaging and chemo-photothermal cancer therapy. Therefore, this work brings new insights for the synthesis of multifunctional nanomaterials and extends theranostic applications.

Optical second-harmonic generation imaging for identifying gastrointestinal stromal tumors

Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors arising in the digest tract.It brings a challenge to diagnosis because it is asymptomatic clinically.It is well known that tumor development is often accompanied by the changes in the morphology of collagen fibers.Nowadays,an emerging optical imaging technique,second-harmonic generation(SHG),can directly identify collagen fibers without staining due to its noncentrosymmetric properties.Therefore,in this study,we attempt to assess the feasibility of SHG imaging for detecting GISTs by monitoring the morphological changes of collagen fibers in tumor microenvironment.We found that collagen alterations occurred obviously in the GISTs by comparing with normal tissues,and furthermore,two morphological features from SHG images were extracted to quantitatively assess the morphological difference of collagen fibers between normal muscular layer and GISTs by means of automated image analysis.Quantitative analyses show a significant difference in the two collagen features.This study demonstrates the potential of SHG imaging as an adjunctive diagnostic tool for label-free identification of GISTs.

Monitoring microenvironment of Hep G2 cell apoptosis using two-photon fluorescence lifetime imaging microscopy

Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.

Simultaneous Measurement of Neural Activities of Acute Mouse Hippocampal Slices Using Multi-Electrode Array System and Laser Confocal Calcium Imaging

Recently, non-invasive, real-time and multi-point measurement of neural activities has become possible by using a multi-electrode array (MEA). Another method for multi-point measurement is the fluorescent imaging technique using voltage indicator dyes or calcium indicator dyes. Especially, calcium imaging using fluorescent calcium indicator dyes is often more useful, because they exhibit larger changes in the fluorescence intensity than voltage indicator dyes and their fluorescence changes can be detect easily. Additionally, calcium signals play key roles in the brain function, such as the long-term potentiation (LTP) in the hippocampus, and calcium imaging can be a powerful tool to elucidate the brain function. In this study, we constructed a measurement apparatus combining the MEA system and laser confocal calcium imaging and simultaneously measured electric signals and calcium signals in acute mouse hippocampal slices. The obtained results showed the availability of the present method.

Synthesis of fluorescent calcium phosphate/carbon dot hybrid composites for detection of copper ions and cell imaging

As a new form of fluorescent carbon nanomaterials, carbon dots （CDs） have many advantages such as simplicity of synthesis, low biological toxicity, resistance to photobleaching, excellent in biocompatibility and easy to modify. As the most inorganic mineral of human hard tissues including bone and tooth, calcium phosphate （CaP） materials have high biocompatibility and good biodegradability. The hydroxyapatite （HA）, a common type of calcium phosphate, has been widely used in drug delivery, removal of heavy metal ions, gene transfection and other biomedical fields. Herein, a novel method for preparation of fluorescent calcium phosphate/carbon dot （ CaP/ CD） hybrid composites was described. We first prepared CDs with a quantum yield of 51.5 % by carbonization of citric acid with polyethylenimine （PEI） in one step. Next, the fluorescent CaP/CD hybrid composites upon UV ex- citation were obtained in the presence of carboxymethyl cellulose （CMC） as a template and CD as a co-template. The as-prepared CaP/CD hybrid composites consisted of irregularly shaped nanorods with a length of 50 - 140 nm and a diameter of 10- 25 nm were characterized by transmitted electron microscopy （TEM）. And the highly mag- nified TEM image of CaP/CD hybrid composites showed that tens of CDs in shape of nanodot indeed existed in the CaP/CD hybrid composites. More importantly, CDs enabled the CaP/CD hybrid composites to emit bright blue flu- orescence under UV irradiation. In addition, the result of X-ray powder diffraction （XRD） patterns indicated that the crystalline phase of CaP/CD hybrid composites were in good agreement with those of hexagonal HA （JCPDS Da- ta Card 09-0432）. Moreover, it was found that the as-synthesized CaP/CD hybrid composites were potentially use- ful in detecting the concentration of Cu2＋ and imaging the cell with nontoxic as potential fluorescent probes.

Zebrafish imaging and two-photon fluorescence imaging using ZnSe quantum dots

This study is to report a ZnSe quantum dot with a large two-photon absorption cross section and good biocompatibility,which can be used in bioimaging.Fluorescence emission at 410 nm is observed in the quantum dot under 760-nm laser excitation.These biocompatible quantum dots exhibit a two-photon cross-section of 9.1×105 GM(1 GM=10-50 cm4·s/photon).Two-photon excited laser scanning microscopic images show that cells co-cultured with ZnSe quantum dots are found in the blue channel at a fluorescence intensity that is 14.5 times that of control cells not cocultured with quantum dots.After incubating zebrafish larvae with ZnSe quantum dots for 24 h,the fluorescence intensity of the yolk sac stimulated by ultraviolet light is 2.9 times that of the control group.The proposed material shows a great potential application in biological imaging.

Synthesis, Crystal Structure, Two-photon Absorption and Biological Imaging Application of a Water Soluble Carbazole Quaternary Ammonium Compound

A novel carbazole quaternary ammonium compound（abbreviated as T_2） had been synthesized and characterized by ~1H NMR, ^（13）C NMR and Mass spectrometry. The single-crystal structure has been determined by X-ray single-crystal diffraction. The electrochemical and two-photon absorption properties of T_2 were systematically studied by cyclic voltammetry and Z-scan determination methods, respectively. The results suggested that T_2 had a good oxidation-reduction and excellent nonlinear optical property. The two-photon absorption（TPA） value has a maximum corresponding to cross section σ = 7963.3 GM（Goeppert-Mayer units） at 700 nm, indicating potential applications in nonlinear optical materials. Furthermore, attributing to the excellent water solubility and low cytotoxicity, the compound was explored on its primary application in biological imaging.

AB010. The effect of visual conditioning on cortical map plasticity:a wide-field calcium imaging study

Background:Visual conditioning can refine the response of neurons in the visual cortex and higher visual and cognitive processing of a presented stimulus.This process results in increased sensitivity for that stimulus.The development of new optical imaging technology in the field of neuroscience has led to important advances,notably to better define the functional organization and plasticity of visual areas.The objective of this project is to determine the effect of daily visual conditioning with an oblique sinusoidal grating on the distribution and amplitude of cortical responses.For this,we use wide-field calcium imaging on awake mice,allowing for the observation of responses to a stimulus throughout the entire cortex in real time.Methods:C57BL/6 mice,expressing the GCaMP6s calcium reporter gene,are used to longitudinally measure neuronal activity via wide-field calcium imaging.Spontaneous activity at rest,as well as cortical responses to visual stimuli consisting of sinusoidal networks with orientation(0,30°,60°and 90°),spatial frequency(0.03,0.12,0.24 and 0.48 cpd)and contrast(100%,75%and 50%)variables are recorded to establish cortical maps,as well as tuning curves.Subsequently,the baseline function of the cortex,as well as the cortical representation of visual stimulation(30°or 90°,0.03 cpd and a contrast of 50%,75%and 100%)are studied in the animal before,during,and after daily monocular conditioning,consisting of a specific sinusoidal network(30°,0.03 cpd and 100%)over a period of 7 days.The variations in intensity and activation specificity of various visual cortical areas are calculated according to the visual conditioning and compared to an orientation stimulus for which the animal has not been conditioned(90°).Results:The cortical activation curves show a greater sensitivity of response for stimuli having horizontal or vertical gratings(0 and 90°)than for oblique gratings(30°and 60°)at low spatial frequencies(0,0.3 and 0.12 cpd).However,this trend does not occur with high spatial frequencies(0.24 and 0.48 cpd).Finally,although the intensity of activation varies in a way that is not proportional to the contrast of the stimulation,it would have no influence on the perception of the orientation of the stimuli.Conditioning at a 30°stimulus results in greater activation of the primary visual cortex and some extra-striate visual areas,as well as greater amplification of the ipsilateral cortical responses to the presentation of the visual stimuli.Conclusions:In conclusion,the results demonstrate that visual conditioning would allow for plasticity and consolidation of higher visual pathways.

Perspective of Calcium Imaging Technology Applied to Acupuncture Research

Acupuncture, a therapeutic treatment defined as the insertion of needles into the body at specific points(ie, acupoints), has growing in popularity world-wide to treat various diseases effectively, especially acute and chronic pain. In parallel, interest in the physiological mechanisms underlying acupuncture analgesia, particularly the neural mechanisms have been increasing. Over the past decades, our understanding of how the central nervous system and peripheral nervous system process signals induced by acupuncture has developed rapidly by using electrophysiological methods. However, with the development of neuroscience, electrophysiology is being challenged by calcium imaging in view field, neuron population and visualization in vivo. Owing to the outstanding spatial resolution, the novel imaging approaches provide opportunities to enrich our knowledge about the neurophysiological mechanisms of acupuncture analgesia at subcellular, cellular, and circuit levels in combination with new labeling, genetic and circuit tracing techniques. Therefore, this review will introduce the principle and the method of calcium imaging applied to acupuncture research. We will also review the current findings in pain research using calcium imaging from in vitro to in vivo experiments and discuss the potential methodological considerations in studying acupuncture analgesia.

Highly crystallization-induced emissive luminophores with mechanoluminescent features for two-photon harvesting fluorescence imaging and latent fingerprint identification

Fluorescence imaging can be employed in fields of medical treatment,astronomical exploration,and national defense security.Traditional fluorescence imaging often takes the single-photon techniques,which is vulnerable to background interference and photobleaching.Remedially,two-photon fluorescence imaging can achieve much higher-resolution fluorescence imaging for reducing scattering and deeper depth.Hence,by assembling the tetraphenylethylene backbones with nontoxic and non-noble K^(+)ions,compound 1([(Hdma)K(H_(2)ettc)]_(n),H_(4)ettc=4',4''',4''''',4'''''''-(ethene-1,1,2,2-tetrayl)tetrakis(([1,1'-biphenyl]-4-carboxylic acid)))with the crystallization-induced emissions exhibited charming fluorescence imaging under two-photon excitation microscopy(TPEM).Besides,luminescent powders based on compound 1 can achieve high-resolution fingerprint recognition,providing secure access control and identification for a novel authentication method.Compared with the commercial fluorescent dyes coumarin-6,the as-synthesized compound 1 showed great solvent stability,indicating its durability against harsh environment.Moreover,compound 1 shows mechanoluminescent properties for the perturbation of weak supramolecular interactions within ordered arrangements of the H_(2)ettc^(2−)ligands.This novel compound has provided an important insight to the development of twophoton fluorescence imaging and advanced external-stimuli responsive materials.

Iterative multi-photon adaptive compensation technique for deep tissue two-photon fluorescence lifetime imaging

Fluorescence lifetime imaging can reveal the high-resolution structure of various biophysical and chemical parameters in a microenvironment quantitatively.However,the depth of imaging is generally limited to hundreds of micrometers due to aberration and light scattering in biological tissues.This paper introduces an iterative multi-photon adaptive compensation technique(IMPACT)into a two-photon fluorescence lifetime microscopy system to successfully overcome aberrations and multiple scattering problems in deep tissues.It shows that 400 correction modes can be achieved within 5 min,which was mainly limited by the frame rate of a spatial light modulator.This system was used for high-resolution imaging of mice brain tissue and live zebrafish,further verifying its superior performance in imaging quality and photon accumulation speed.

In situ Raman imaging of high-temperature solid-state reactions in the CaSO4-SiO2 system

The deposition of mineral phases on the heat transfer surfaces of brown coal power plants may have a negative effect on power plant boilers. The paragenesis of these deposits contains information about the actual temperature prevailed during the combustion of lignite, if the temperature-dependences of distinct mineral transformations or reactions are known. Here, we report results of a sintering study (to ?1100℃) with samples containing anhydrite, quartz, and gehlenite, which are typical components of Rhenish lignite ashes. Thermal decompositions and solid-state reactions were analyzed (1) in situ and (2) both in situ and after quenching using confocal hyperspectral Raman imaging. This novel application of confocal Raman spectroscopy provides temperature-and time-resolved, 2-dimensional information about sintering processes with a micrometer-scale resolution. In the course of the sintering experiments with anhydrite and quartz with a weight ratio of 2:1 both polymorphs wollastonite and pseudowollastonite were identified in situ at about 920 and 1000℃, respectively. The formation of pseudowollastonite was thus observed about 120℃ below the phase transition temperature, demonstrating that it can form metastably. In addition,α′L-Ca2SiO4 was identified at about 1100℃. In samples containing equal weight fractions of anhydrite and quartz that were quenched after firing for 9h at about 1100℃,β-Ca2SiO4 (larnite) crystallized as rims around anhydrite grains and in direct contact to wollastonite. We furthermore observed that, depending on the ratio between quartz and anhydrite, wollastonite replaced quartz grains between 920 and 1100℃., i.e., the higher the quartz content, the lower the formation temperature of wollastonite.

PHOTOSWITCHABLE NANOFLUOROPHORES FOR INNOVATIVE BIOIMAGING

Photosensitive fluorescent probes have become powerful tools in chemical biology and molecular biophysics,which are used to investigate cellular processes with high temporal and spatial resolution.Accordingly,photosensitive fluorescent probes,including photoactivatable,photoconvertible,and photoswitchable fluorophores,have been extensively developed during the past decade.The photoswitchable fluorophores have received much attention because they highlight cellular events clearly.This minireview summarizes recent advances of using reversibly photoswitchable fluorophores and their applications in innovative bioimaging.Photoswitchable fluorophores include photoswitchable fluorescent proteins,photoswitchable fluorescent organic molecules(dyes),and photoswitchable fluorescent nanoparticles.Several strategies have been developed to synthesize photoswitchable fluorophores,including engineering combination proteins,chemical synthesis,polymerization,and self-assembly.Here we concentrate on polymer nanoparticles with optically switchable emission properties:either fluorescence on/offor dualalternating-color fluorescence photoswitching.The essential mechanisms of fluorescence photoswitching enable different types of photoswitchable fluorophores to change emission intensity or wavelength(color)and thus validating the basis of the fluorescence on/offor dual-color photoswitching design.Generally the possible applications of any fluorophores are to label biological targets,followed by specific imaging.The newly developed photoswitchable fluorophores enable super-resolution fluorescence imaging because of their photosensitive emission.Finally,we summarize the important area regarding future research and development on photoswitchable fluorescent nanoparticles.

Reperfusion injury components and manifestations determined by cardiovascular MR and MDCT imaging

Advances in magnetic resonance(MR) and computed tomography(CT) imaging have improved visualization of acute and scar infarct.Over the past decade,there have been and continues to be many significant technical advancements in cardiac MR and multi-detector computed tomography(MDCT) technologies.The strength of MR imaging relies on a variety of pulse sequences and the ability to noninvasively provide information on myocardial structure,function and perfusion in a single imaging session.The recent technical developments may also allow CT technologies to rise to the forefront for evaluating clinical ischemic heart disease.Components of reperfusion injury including myocardial edema,hemorrhage,calcium deposition and microvascular obstruction(MO) have been demonstrated using MR and CT technologies.MR imaging can be used serially and noninvasively in assessing acute and chronic consequences of reperfusion injury because there is no radiation exposure or administration of radioactive materials.MDCT is better suited for assessing coronary artery stenosis and as an alternative technique for as-sessing viability in patients where MR imaging is contraindicated.Changes in left ventricular(LV) volumes and function measured on cine MR are directly related to infarct size measured on delayed contrast enhanced images.Recent MR studies found that transmural infarct,MO and peri-infarct zone are excellent predictors of poor post-infarct recovery and mortality.Recent MR studies provided ample evidence that growth factor genes and stem cells delivered locally have beneficial effects on myocardial viability,perfusion and function.The significance of deposited calcium in acute infarct detected on MDCT requires further studies.Cardiac MR and MDCT imaging have the potential for assessing reperfusion injury components and manifestations.