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Objective To build a neural network based on the Unet infrastructure for recognition and segmentation of two-dimensional calcium imaging fluorescence images.Methods The in vivo miniaturized two-photon microscope(mTPM)was used for brain calcium imaging in freely moving mice.The imaging data was motion corrected using the NoRMCorre algorithm and processed using ImageJ software to obtain the original images after correction,and the labels were produced using the Labelme software.The neural network HDCGUnet was built using the original images and labels for training,and optimized to improve the model structure according to the training effect.Finally,the evaluation indexes were selected and compared with those of other models to verify the utility of this model.Results The HDCGUnet model,which was collected and made on our own,performed best in the two-photon calcium imaging dataset compared to other models,and performed well on the BBBC dataset either.Conclusion The HDCGUnet model provides a novel alternative for the recognition and segmentation of two-photon calcium imaging images.
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The perception of motion is an important function of vision. Neural wiring diagrams for extracting directional information have been obtained by connectome reconstruction. Direction selectivity in Drosophila is thought to originate in T4/T5 neurons through integrating inputs with different temporal filtering properties. Through genetic screening based on synaptic distribution, we isolated a new type of TmY neuron, termed TmY-ds, that form reciprocal synaptic connections with T4/T5 neurons. Its neurites responded to grating motion along the four cardinal directions and showed a variety of direction selectivity. Intriguingly, its direction selectivity originated from temporal filtering neurons rather than T4/T5. Genetic silencing and activation experiments showed that TmY-ds neurons are functionally upstream of T4/T5. Our results suggest that direction selectivity is generated in a tripartite circuit formed among these three neurons-temporal filtering, TmY-ds, and T4/T5 neurons, in which TmY-ds plays a role in the enhancement of direction selectivity in T4/T5 neurons.
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Animales , Neuritas , Drosophila , Neuronas , ConectomaRESUMEN
@#The development of intravital microscopy (IVM) has enabled researchers to perform in-situ, real-time observations of pulmonary micro-circulation at the cellular level, and has become instrumental for researching the immune micro-environment of pulmonary diseases. This article introduces the developments in constructing the pulmonary imaging window and summarizes the current light microscopy techniques used for lung IVM with regard to its relevant functional and application features, which includes wide field fluorescence microscopy, confocal microscopy, as well as two-photon microscopy. It then provides examples of IVM of pulmonary immune response in inflammation and infection in murine models, and finally specifies the technological limitations to provide reference for researchers to systematically learn and understand the technology.
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The glymphatic system is a fluid dynamics network that is important for maintaining homeostasis of the brain, and it is also a new target for the treatment of various central nervous system diseases. The crucial point regarding research into the glymphatic system is the microhydrodynamics of the cerebrospinal fluid tracer. This review summarizes the emerging technologies, such as magnetic resonance technology, two photon microscopic imaging technology, near infrared fluorescence imaging technology, and transcranial macroscopic imaging, and summarizes its research applications and technical advantages to provide methodological strategies for basic and clinical research on glymphatic system function.
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Objective To establish a method of in vivo microvascular imaging and blood flow calculation with better continuous imaging quality. Methods Anesthetized mice with dye injection through tail vein were placed in the center of a 37 ℃ hot plate holder. The stripped tissues were placed in a self￣made low edge confocal dish and immersed in normal saline. The exposed tissues were pressed with self￣made circular metal pads of different weights and sizes. The microvascular blood flow in the liver and hind limb of pregnant mice (n = 3) and normal female mice (n = 3) were measured by the improved device. Results This method can accomplish stable and continuous imaging. The blood flow velocity of liver and hind limb of pregnant mice were faster than that of normal female mice. Conclusion This method can be used for three￣dimensional imaging of microvessels and detection of blood flow velocity in organs.
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Objective:To evaluate the skin development and repair process of X-ray radiation damage in rat with non-invasive two-photon excitation fluorescence (TPEF) imaging technology in vivo. Methods:Totally 24 SD rats were randomly divided into four groups including X-ray irradiated group (25, 35 and 45 Gy) and non-irradiation control group. At different times after irradiation, the degree of skin injury was evaluated, and the pathological changes of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and collagen fiber fluorescence signals in epidermal cells were detected in vivo by TPEF imaging technology. Results:At 10 d post-irradiation, the skin of irradiation groups showed erythema and desquamation. At 15-20 d post-irradiation, the skin of radiation groups developed progressive exudation, edema and ulcers with increasing radiation dose. On day 25, the skin began to repair in the 25 Gy group, however, the skin of other groups still had exudation and ulcers. On day 10, NAD(P)H fluorescence signal in epidermal cells of irradiation groups decreased and the fluorescence signal of collagen fibers in papillary layer and reticular layer of irradiation groups reduced, which were significantly lower than that of normal control group ( t=24.145, 28.303, 26.989, 6.654, 7.510, 7.997, P<0.05). On day 30, fluorescence signal of NAD(P)H and collagen fibers in epidermal cells and dermis began to repair, the cell from stratum granulosum, stratum spinosum, and stratum basale in the 25 Gy group showed fluorescence signal, the other groups did not show. The fluorescence signal of collagen fibers in the 25 Gy group were gradually increased in papillary layer and reticular layer, however, they were significantly lower than normal control group ( t=115.133, 17.431, P<0.05), the skin of 45 Gy group did not show fluorescence signal of collagen fibers. Conclusions:The damage and repair process of epidermal cells and dermal collagen fiber can be detected noninvasively by TPEF imaging technology after X-ray irradiation in vivo.
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@#Calcium is an important messenger in the mammalian nerve cells which mediates a variety of intracellular signal transduction pathways and plays critical roles in regulating the neuronal functions. Calcium signaling exerts its highly specific function in a defined sub-region of the cell, especially in the visual cortex of the brain. Detection of calcium signals in neurons is particularly important for the studying of neuronal function. The two-photon microscope has a unique advantage in the detection of calcium signal in the superficial cortex. In this paper, the application of two-photon in the <i>in vivo</i> detection of the visual cortical Ⅱ/Ⅲ layer of model animals are reviewed.
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An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular, cellular, circuit, and system levels. The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors. In this brief review, we summarize recent advances in miniature fluorescence microscopy for neuroscience, focusing mostly on two mainstream solutions - miniature single-photon microscopy, and miniature two-photon microscopy. We discuss their technical advantages and limitations as well as unmet challenges for future improvement. Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements, long and complex protocols, and even disease progression and aging.
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Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca imaging. Notably, we were able to record single-spine spontaneous Ca activity in specific circuits. Distinct spontaneous Ca dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits. Our method can be applied to monitor Ca dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.
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An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular, cellular, circuit, and system levels. The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors. In this brief review, we summarize recent advances in miniature fluorescence microscopy for neuroscience, focusing mostly on two mainstream solutions - miniature single-photon microscopy, and miniature two-photon microscopy. We discuss their technical advantages and limitations as well as unmet challenges for future improvement. Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements, long and complex protocols, and even disease progression and aging.
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Objective: To evaluate the effect of two-photon in vivo imaging on detecting the blood brain barrier (BBB) injury in the ultra-early stage of cerebral ischemic stroke. Methods: Twelve clean grade C57BL/6 healthy male mice aged 8-12 weeks were randomly divided into Sham group and middle cerebral artery occlusion (MCAO) group,which were sham operated or middle cerebral artery occluded,respectively. After 60 min of ischemia,MCAO mice were treated with reperfusion for 30-60 min after the suture being removed. The vessels and the neutrophils of mice in the two groups were labeled intravenously with Alexa-Fluora-488 conjugated dextran and rhodamine 6G,respectively. The integrity of BBB was detected by intravenous injection of tetramethylrhodamine-5-maleimide (TMR). Before and after the stroke,the real-time changes of the fluorescence intensity of the inside and outside cerebral vessels of mice in the MCAO group were observed by two-photon fluorescence microscopy. Results: The fluorescence intensity of TMR in the external cerebrovascular of mice in the MACO group was significantly increased within 30-60 min after stroke (P=0.000),suggesting there existed tracer leakage. Compared with the Sham group,the movement of neutrophils in the blood vessels of mice in the MACO group was significantly slowed down (P=0.000). Conclusion: Two-photon in vivo imaging can be used to detect the BBB injury in the ultra-early stage of cerebral ischemic stroke,which provides a certain reference value for clinical application.
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Objective Due to the distortion of hepatic cells in hepatic steatosis, the characteristics of blood flow in the liver could change. This study observed the morphology, blood flow velocity and tortuosity changes aimed to help the diagnosis and treatment in the hepatic steatosis. Methods The hepatic steatosis model was established by subcutaneous injection of carbon tetrachloride (CCl4) and olive oil in mice, and then liver tissue was stained with HE and oil red 0 staining. Laser ultrasound was used to measure the blood flow changes in the superficial hepatic vessels of the left lobe. The mice's tail veins were injected with Texas red fluorescent dye, then two-photon fluorescence microscopy were used to detect the flow of red blood cells in mice's hepatic sinusoids, blood vessel diameter, the hepatic sinusoidal tortuosity. Results After injected with CCl4 for two(n= 16) or four(n= 16) weeks, the oil red 0 staining indicated lipid accumulation in hepatic cells, especially around the central vein. HE staining indicated narrowing of the hepatic sinusoidal vessels, and more obviously in 4-weeks group. As the modeling time increased, the blood flow velocity decreased gradually in hepatic sinusoids and superficial hepatic vessels in the left lobe, and the diameter of the hepatic sinusoids became smaller. Conclusion In the hepatic steatosis, the internal diameter of hepatic sinus decreases, and the blood flow also decreases in the hepatic sinusoids, but hepatic sinusoidal tortuosity increases. All of this provide a visual morphological experimental basis for the early diagnosis and treatment to the hepatic steatosis.
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Photodynamic therapy (PDT) is one of the new approaches for cancer treatment with high efficacy. However, applications of current photosensitizers are restricted to skin and superficial tumor due to poor in vivo targeting ability, poor water solubility and short wavelength excitement, which limits penetration therefore therapeutic depth. Here, a biodegradable polymeric micelle, methoxy poly(ethylene glycol)-polylactide copolymer (mPEG-PDLLA), is employed as drug delivery system to co-encapsulate strong two-photon absorption compound (LTPA) and photosensitizers. This delivery system is designed to target tumor passively, resulting in near infrared light with an approximately 808 nm wavelength becoming able to indirectly excite photosensitizers through fluorescence resonance energy transfer. Tumor cells and microvessels could be damaged by the generated singlet oxygen. The average size of drug loaded micelles was approximately 55 nm and showed a spherical shape. Both compounds could be released simultaneously from micelles under either weak acid and neutral pH conditions. Reactive oxygen species was produced intracellularly during two-photon PDT process and induced cell apoptosis/necrosis, which was quantified by Annexin-V/FITC assays. Time-dependent ex vivo organ distribution and in vivo anticancer efficacy results suggested that the drug carriers could accumulate in tumors and suppress tumor growth by two-photon PDT. All animals experiments were performed in line with national regulations and approved by the Animal Experiments Ethical Committee of College of Pharmaceutical Sciences, Southwest University. In summary, we have employed two-photon PDT for breast cancer treatment successfully in a mouse model and have demonstrated the significance of delivery system in such therapeutics.
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Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.
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Absorción , Enfermedades Transmisibles , Flavina-Adenina Dinucleótido , Fluorescencia , Microscopía Fluorescente , NAD , Fotones , Triptófano , Cicatrización de HeridasRESUMEN
Objective To compare the removal efficiency of γδT cells between cornea and ear skin and develop an alternative method for dynamic monitoring of γδT cells in mouse cornea in vivo using 2-photon laser scanning microscopy.Methods The γδT cells in mouse ear skin were monitored before and after antibody neutralization,and the mice corneas were excised and stained for counting γδT cells at 6 h,12 h,24 h after antibody neutralization by using 2-photon laser scanning microscopy,followed by comparison of the removal efficiency of γδT cells between the cornea and ear skin.Results The γδT cells in normal mouse cornea were often distributed in the limbal epithelium and superficial stromal layer.The irregular morphology of γδT cells in the epithelial layer was often accompanied by protuberances,while the stromal γδT cells were mostly round or oval and the number of cells was approximately 27 ± 4.After antibody neutralization,the number of γδT cells in the cornea of mice gradually decreased,and the number of cells at 6 h,12 h and 24 h was significantly lower than that of before depletion (P =0.03,0.00,0.00),and the removal efficiencies were 48％,78％,and 96％,respectively.The γδT cells in ear skin of the normal mice were ellipse or stellate with cell processes and they were located in epidermal layer,and the cell number was about 60 ± 9.After antibody neutralization,the number of γδT cells were significantly reduced at 6 h,12 h and 24 h compared with before depletion (P =0.000,0.000,0.000) and the removal efficiency were 43％,72％ and 95％,respectively.Conclusion The number of γδT cells in the cornea and ear skin is gradually decreased after antibody neutralization,and their removal efficiency is consistent with time.Therefore,monitoring the γδT cells in the mouse ear skin is an ideal alternative to dynamically monitoring the changes in the number of γδT cells in the cornea in vivo.
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Photodynamic therapy (PDT) has emerged as a more effective and promising treatment towards cancer therapy. PDT is a minimally invasive and spatially selective medical technique to destroy cancer cells without drug resistance, which has been increasingly applied in the clinical praxis alongside surgery, chemotherapy and radiotherapy. However, traditional PDTs use a high energy one-photon laser beam, which is far from the efficient optical window of mammalian tissue (650−950 nm). Moreover, it has great limitations in the depth of penetration, and induces the undesired light toxicity. The development of photosensitizers has always been a bottleneck to the effective application of PDT in clinical practice. From the first generation of hematoporphyrin derivatives to the third-generation photosensitizers with tumor targeting ability, they meet the urgent clinical needs to some extent, but they still can not satisfy the requirements of two-photon PDT. Therefore, the development of photosensitizers, which are capable of two-photon activated PDT, has become a promising approach. Among the various two-photon absorption photosensitizers, ruthenium (Ⅱ) polypyridyl complexes have been recognized as excellent candidates due to their attractive photophysical properties. This review is prepared to summarize the recent achievements in the application of ruthenium (Ⅱ) polypyridyl complexes as photosensitizers for two-photon PDT, as well as to provide guidance for the design of two-photon activated photosensitizers in future research.
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@#Twophoton microscopy is a new technique which combines laser scanning con-focal microscopy and two-photon excitation technique. Two-photon fluorescence microscopy has the advantages of little light damage, small bleaching area, strong penetrability, high resolution, high fluorescence collection efficiency, and high image contrast. It is suitable for dark field imaging and multi-labeled compound measurement, and has been widely used in small animals in vivo optical imaging, such as research for tumour, gene therapy, stem cells, drug development, spinal cord injury, etc.
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In the central nervous system, ionotropic AMPA receptors mediate the majority of the fast excitatory synaptic transmission.Trafficking of AMPA receptors into and out of synapses is a highly dynamic process, which plays a key role in synaptic plasticity that is critical for higher brain functions such as learning and memory. Genetic alterations in AMPA receptor or proteins that regulate AMPA receptors trafficking have been implicated in various diseases, including autism spectrum disorders, schizophrenia, Alzheimer′s disease, and intellectual disability. Thus, elucidating the regulation of AMPA trafficking and function is vital to understanding higher brain functions. Many studies have used live imaging of fluorescently tagged AMPA receptors to directly monitor their membrane trafficking in real time,but most of these studies were performed in vitro using neuronal cell cultures or brain slices.Recent technological advances have allowed the imaging of synaptic proteins in vivo in intact organisms, which enables the visualization of synaptic plasticity at a molecular level in living animals.In this review, we mainly discuss the latest development in AMPA receptors trafficking studies and elucidate the contributions of receptor imaging in vitro and in vivo to understanding the molecular mechanisms under-lying synaptic plasticity.
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OBJECTIVE: To prepare a kind of novel multifunctional gold nanoshell coated magnetic nanoparticles and investigate their morphology, size distribution, two-photon optical properties, cellular uptake behavior, cytotoxicity as well as drug release profile. METHODS: In this study, the gold shell coated magnetic polyester nanoparticles (AuMPn) were prepared by emulsion solvent evaporation method combined with hydroxylamine reduction method. The size distribution, morphology, and two-photon optical properties were characterized by Zeta sizer, AFM, TEM and multiphoton confocal laser scanning microscopy respectively. The drug loading and release profile were evaluated by dialysis method using idarubicin as a model drug. RESULTS: The resultant AuMPn exhibit spherical shaped morphology with average size of (235.7±1.34) nm. The encapsulation efficiency and drug loading efficiency were 72.08% and 1.73% respectively. Under near infrared light irradiation, gold shell outside of AuMPn showed fingerprint like characteristic emission spectrum. The in vitro drug release study demonstrated that the release of idarubicin from AuMPn can be controlled by NIR irradiation. CONCLUSION: Overall, a novel gold shell coated nanoparticles are successfully prepared in this study. By taking advantage of the dual function of the gold shell outside of the particles, precise control on drug release as well as label-free visualization of AuMPn can be achieved simultaneously. Thus, this approach provides a new strategy for drug delivery.
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Damage in the periphery or spinal cord induces maladaptive plastic changes along the somatosensory nervous system from the periphery to the cortex, often leading to chronic pain. Although the role of neural circuit remodeling and structural synaptic plasticity in the 'pain matrix' cortices in chronic pain has been thought as a secondary epiphenomenon to altered nociceptive signaling in the spinal cord, progress in whole brain imaging studies on human patients and animal models has suggested a possibility that plastic changes in cortical neural circuits may actively contribute to chronic pain symptoms. Furthermore, recent development in two-photon microscopy and fluorescence labeling techniques have enabled us to longitudinally trace the structural and functional changes in local circuits, single neurons and even individual synapses in the brain of living animals. These technical advances has started to reveal that cortical structural remodeling following tissue or nerve damage could rapidly occur within days, which are temporally correlated with functional plasticity of cortical circuits as well as the development and maintenance of chronic pain behavior, thereby modifying the previous concept that it takes much longer periods (e.g. months or years). In this review, we discuss the relation of neural circuit plasticity in the 'pain matrix' cortices, such as the anterior cingulate cortex, prefrontal cortex and primary somatosensory cortex, with chronic pain. We also introduce how to apply long-term in vivo two-photon imaging approaches for the study of pathophysiological mechanisms of chronic pain.