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A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.
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Conducta Animal , Peces/fisiología , Animales , Microscopía/métodos , Neuronas/fisiología , Natación/fisiologíaRESUMEN
Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 µm laterally and 3.35 µm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.
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Conducta Animal , Encéfalo/diagnóstico por imagen , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Miniaturización/métodos , Animales , Encéfalo/citología , Encéfalo/fisiología , Técnicas Citológicas/métodos , RatonesRESUMEN
The loss-of-function mutation in PARK7/DJ-1 is one of the most common causes of autosomal recessive Parkinson's disease, and patients carrying PARK7 mutations often exhibit both a progressive movement disorder and emotional impairment, such as anxiety. However, the causes of the emotional symptom accompanying PARK7-associated and other forms of Parkinson's disease remain largely unexplored. Using two-photon microscopic Ca2+ imaging in awake PARK7-/- and PARK7+/+ mice, we found that (i) PARK7-/- neurons in the frontal association cortex showed substantially higher circuit activity recorded as spontaneous somatic Ca2+ signals; (ii) both basal and evoked dopamine release remained intact, as determined by both electrochemical dopamine recordings and high performance liquid chromatography in vivo; (iii) D2 receptor expression was significantly decreased in postsynaptic frontal association cortical neurons, and the hyper-neuronal activity were rescued by D2 receptor intervention using either local pharmacology or viral D2 receptor over-expression; and (iv) PARK7-/- mice showed anxiety-like behaviours that were rescued by either local D2 receptor pharmacology or overexpression. Thus, for first time, we demonstrated a robust D2 receptor-dependent phenotype of individual neurons within the prefrontal cortex circuit in awake parkinsonian mice that linked with anxiety. Our work sheds light on early-onset phenotypes and the mechanisms underlying Parkinson's disease by imaging brain circuits in an awake mouse model.
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Enfermedad de Parkinson/genética , Enfermedad de Parkinson/fisiopatología , Proteína Desglicasa DJ-1/genética , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Femenino , Humanos , Masculino , Ratones , Enfermedad de Parkinson/metabolismo , Trastornos Parkinsonianos/metabolismo , Corteza Prefrontal/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Sustancia Negra/metabolismo , VigiliaRESUMEN
The ability of the brain to predict future events based on the pattern of recent sensory experience is critical for guiding animal's behavior. Neocortical circuits for ongoing processing of sensory stimuli are extensively studied, but their contributions to the anticipation of upcoming sensory stimuli remain less understood. We, therefore, used in vivo cellular imaging and fiber photometry to record mouse primary auditory cortex to elucidate its role in processing anticipated stimulation. We found neuronal ensembles in layers 2/3, 4, and 5 which were activated in relationship to anticipated sound events following rhythmic stimulation. These neuronal activities correlated with the occurrence of anticipatory motor responses in an auditory learning task. Optogenetic manipulation experiments revealed an essential role of such neuronal activities in producing the anticipatory behavior. These results strongly suggest that the neural circuits of primary sensory cortex are critical for coding predictive information and transforming it into anticipatory motor behavior.
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Corteza Auditiva/fisiología , Percepción Auditiva/fisiología , Motivación/fisiología , Actividad Motora/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Potenciales de Acción/fisiología , Animales , Corteza Auditiva/citología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Condicionamiento Clásico , Conducta de Ingestión de Líquido , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Parvalbúminas/genética , Parvalbúminas/metabolismo , Transducción Genética , VigiliaRESUMEN
In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd=561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to -900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.
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Calcio/metabolismo , Fluorometría/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuroimagen/métodos , Potenciales de Acción/fisiología , Animales , Calcio/análisis , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Dendritas/metabolismo , Dendritas/fisiología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Ácido Egtácico/metabolismo , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND/AIMS: Obesity and high salt intake are major risk factors for hypertension and cardiometabolic diseases. Obese individuals often consume more dietary salt. We aim to examine the neurophysiologic effects underlying obesity-related high salt intake. METHODS: A multi-center, random-order, double-blind taste study, SATIETY-1, was conducted in the communities of four cities in China; and an interventional study was also performed in the local community of Chongqing, using brain positron emission tomography/computed tomography (PET/CT) scanning. RESULTS: We showed that overweight/obese individuals were prone to consume a higher daily salt intake (2.0 g/day higher compared with normal weight individuals after multivariable adjustment, 95% CI, 1.2-2.8 g/day, P < 0.001), furthermore they exhibited reduced salt sensitivity and a higher salt preference. The altered salty taste and salty preference in the overweight/obese individuals was related to increased activity in brain regions that included the orbitofrontal cortex (OFC, r = 0.44, P= 0.01), insula (r = 0.38, P= 0.03), and parahippocampus (r = 0.37, P= 0.04). CONCLUSION: Increased salt intake among overweight/obese individuals is associated with altered salt sensitivity and preference that related to the abnormal activity of gustatory cortex. This study provides insights for reducing salt intake by modifying neural processing of salty preference in obesity.
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Obesidad/fisiopatología , Sobrepeso/fisiopatología , Cloruro de Sodio Dietético/efectos adversos , Gusto/fisiología , Adolescente , Adulto , Anciano , Presión Sanguínea/efectos de los fármacos , Método Doble Ciego , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones , Factores de Riesgo , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Glial precursor transplantation provides a potential therapy for brain disorders. Before its clinical application, experimental evidence needs to indicate that engrafted glial cells are functionally incorporated into the existing circuits and become essential partners of neurons for executing fundamental brain functions. While previous experiments supporting for their functional integration have been obtained under in vitro conditions using slice preparations, in vivo evidence for such integration is still lacking. Here, we utilized in vivo two-photon Ca(2+) imaging along with immunohistochemistry, fluorescent indicator labeling-based axon tracing and correlated light/electron microscopy to analyze the profiles and the functional status of glial precursor cell-derived astrocytes in adult mouse neocortex. We show that after being transplanted into somatosensory cortex, precursor-derived astrocytes are able to survive for more than a year and respond with Ca(2+) signals to sensory stimulation. These sensory-evoked responses are mediated by functionally-expressed nicotinic receptors and newly-established synaptic contacts with the host cholinergic afferents. Our results provide in vivo evidence for a functional integration of transplanted astrocytes into adult mammalian neocortex, representing a proof-of-principle for sensory cortex remodeling through addition of essential neural elements. Moreover, we provide strong support for the use of glial precursor transplantation to understand glia-related neural development in vivo.
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Astrocitos/fisiología , Astrocitos/trasplante , Señalización del Calcio/fisiología , Estimulación Eléctrica/métodos , Neocórtex/citología , Neocórtex/fisiología , Corteza Somatosensorial/fisiología , Potenciales de Acción/fisiología , Animales , Astrocitos/citología , Diferenciación Celular/fisiología , Femenino , Humanos , Masculino , Ratones , Neocórtex/cirugía , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Células-Madre Neurales/trasplante , Transducción de Señal/fisiologíaRESUMEN
Sensory information reaches the cortex through synchronously active thalamic axons, which provide a strong drive to layer 4 (L4) cortical neurons. Because of technical limitations, the dendritic signaling processes underlying the rapid and efficient activation of L4 neurons in vivo remained unknown. Here we introduce an approach that allows the direct monitoring of single dendritic spine Ca(2+) signals in L4 spiny stellate cells of the vibrissal mouse cortex in vivo. Our results demonstrate that activation of N-methyl-D-aspartate (NMDA) receptors is required for sensory-evoked action potential (AP) generation in these neurons. By analyzing NMDA receptor-mediated Ca(2+) signaling, we identify whisker stimulation-evoked large responses in a subset of dendritic spines. These sensory-stimulation-activated spines, representing predominantly thalamo-cortical input sites, were denser at proximal dendritic regions. The amplitude of sensory-evoked spine Ca(2+) signals was independent of the activity of neighboring spines, without evidence for cooperativity. Furthermore, we found that spine Ca(2+) signals evoked by back-propagating APs sum linearly with sensory-evoked synaptic Ca(2+) signals. Thus, our results identify in sensory information-receiving L4 cortical neurons a linear mode of dendritic integration that underlies the rapid and reliable transfer of peripheral signals to the cortical network.
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Señalización del Calcio/fisiología , Corteza Cerebral/metabolismo , Dendritas/metabolismo , Red Nerviosa/fisiología , Médula Espinal/metabolismo , Animales , Corteza Cerebral/citología , Ratones , Receptores de N-Metil-D-Aspartato/metabolismo , Médula Espinal/citologíaRESUMEN
In sensory cortex regions, neurons are tuned to specific stimulus features. For example, in the visual cortex, many neurons fire predominantly in response to moving objects of a preferred orientation. However, the characteristics of the synaptic input that cortical neurons receive to generate their output firing pattern remain unclear. Here we report a novel approach for the visualization and functional mapping of sensory inputs to the dendrites of cortical neurons in vivo. By combining high-speed two-photon imaging with electrophysiological recordings, we identify local subthreshold calcium signals that correspond to orientation-specific synaptic inputs. We find that even inputs that share the same orientation preference are widely distributed throughout the dendritic tree. At the same time, inputs of different orientation preference are interspersed, so that adjacent dendritic segments are tuned to distinct orientations. Thus, orientation-tuned neurons can compute their characteristic firing pattern by integrating spatially distributed synaptic inputs coding for multiple stimulus orientations.
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Dendritas/fisiología , Células Receptoras Sensoriales/fisiología , Corteza Visual/citología , Potenciales de Acción , Animales , Señalización del Calcio , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Células Receptoras Sensoriales/citología , Sinapsis/metabolismoRESUMEN
Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo two-photon calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.
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Señalización del Calcio/fisiología , Dendritas/metabolismo , Corteza Motora/citología , Células Piramidales/metabolismo , Sinapsis/fisiología , Animales , Electroporación , Ratones , Corteza Motora/metabolismo , Técnicas de Placa-ClampRESUMEN
Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons' output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron. Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by single whiskers, but many spines were activated by multiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising from multiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.
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Corteza Cerebral/fisiología , Dendritas/fisiología , Sensación/fisiología , Animales , Calcio/metabolismo , Espinas Dendríticas/fisiología , Estimulación Eléctrica , Ratones , Ratones Endogámicos C57BL , Estimulación Física , Factores de Tiempo , Vibrisas/fisiologíaRESUMEN
Existing stress evaluation methods based on the Lamb waves mainly use the time of flight (TOF) or velocity as the means of stress measurement. However, these two features used for stress measurement are sometimes insensitive to stress changes. Therefore, it is essential to explore other features that are potentially more sensitive to stress changes. The time-frequency spectrums of signals containing stress information have not yet been fully studied for stress evaluation. This paper proposes a uniaxial stress measurement method based on two time-frequency characteristics of Lamb waves, i.e., the slope of time-frequency spectrum distribution (TFSD) and pulse width impact factor. Theoretical expressions of the slope of TFSD are derived. The impacts of excitation signal parameters (i.e., bandwidth and center frequency) and noise on two time-frequency characteristics were discussed. Then, the fitting results of the finite element simulation are consistent with the results predicted by theory. To experimentally validate the proposed theory, aluminum plate specimens with two different types of adhesives were used for the experiment. According to the experimental stress measurement expression, three uniaxial tensile tests in the range of 35-95 MPa were conducted on the identical batch of specimens. The maximum standard deviation of multiple measured stress based on pulse width impact factor is 3.76433 MPa, demonstrating excellent measurement stability. The maximum standard deviation of multiple measured stress based on the slope of TFSD is 9.12492 MPa. It shows that the proposed methodology is a promising alternative for stress measurement.
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In recent years, the number of functional magnetic resonance imaging (fMRI) research in acupuncture grows increasingly. However, due to the differences in acupoint selection, acupuncture technique and sample size, the problems get more prominent in terms of the diverse results and the lack of common rules of acupuncture among researches. By taking the fMRI research for post-stroke motor dysfunction (PSMD) treated with acupuncture as the example, this paper introduces the fMRI Meta-analysis technology for integrating the relevant research results and extracting the common rules, namely image-based Meta-analysis (IBMA) and coordinate-based Meta-analysis (CBMA). Considering the higher feasibility of CBMA, three available CBMA methods are explained specially, including activation likelihood estimation (ALE), kernel density analysis (KDA), and seed-based d mapping (SDM). Focusing on the precautions and operation procedure of CBMA, the review is conducted systematically on the type of fMRI research, task design, analytical method, and the thinking integrity of fMRI Meta-analysis, and the review findings are collated in charts. It aims to assist readers to understand the abstract and complex theories and practical information of this technology efficiently, conveniently and systematically, and hopes to provide the references for the future learning and the application.
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Terapia por Acupuntura , Puntos de Acupuntura , Aprendizaje , Imagen por Resonancia Magnética , Tamaño de la MuestraRESUMEN
Two-photon Ca2+ imaging technology increasingly plays an essential role in neuroscience research. However, the requirement for extensive professional annotation poses a significant challenge to improving the performance of neuron segmentation models. Here, we present NeuroSeg-III, an innovative self-supervised learning approach specifically designed to achieve fast and precise segmentation of neurons in imaging data. This approach consists of two modules: a self-supervised pre-training network and a segmentation network. After pre-training the encoder of the segmentation network via a self-supervised learning method without any annotated data, we only need to fine-tune the segmentation network with a small amount of annotated data. The segmentation network is designed with YOLOv8s, FasterNet, efficient multi-scale attention mechanism (EMA), and bi-directional feature pyramid network (BiFPN), which enhanced the model's segmentation accuracy while reducing the computational cost and parameters. The generalization of our approach was validated across different Ca2+ indicators and scales of imaging data. Significantly, the proposed neuron segmentation approach exhibits exceptional speed and accuracy, surpassing the current state-of-the-art benchmarks when evaluated using a publicly available dataset. The results underscore the effectiveness of NeuroSeg-III, with employing an efficient training strategy tailored for two-photon Ca2+ imaging data and delivering remarkable precision in neuron segmentation.
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Quantitative and mechanistic understanding of learning and long-term memory at the level of single neurons in living brains require highly demanding techniques. A specific need is to precisely label one cell whose firing output property is pinpointed amidst a functionally characterized large population of neurons through the learning process and then investigate the distribution and properties of dendritic inputs. Here, we disseminate an integrated method of daily two-photon neuronal population Ca2+ imaging through an auditory associative learning course, followed by targeted single-cell loose-patch recording and electroporation of plasmid for enhanced chronic Ca2+ imaging of dendritic spines in the targeted cell. Our method provides a unique solution to the demand, opening a solid path toward the hard-cores of how learning and long-term memory are physiologically carried out at the level of single neurons and synapses.
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The development of two-photon microscopy and Ca2+ indicators has enabled the recording of multiscale neuronal activities in vivo and thus advanced the understanding of brain functions. However, it is challenging to perform automatic, accurate, and generalized neuron segmentation when processing a large amount of imaging data. Here, we propose a novel deep-learning-based neural network, termed as NeuroSeg-II, to conduct automatic neuron segmentation for in vivo two-photon Ca2+ imaging data. This network architecture is based on Mask region-based convolutional neural network (R-CNN) but has enhancements of an attention mechanism and modified feature hierarchy modules. We added an attention mechanism module to focus the computation on neuron regions in imaging data. We also enhanced the feature hierarchy to extract feature information at diverse levels. To incorporate both spatial and temporal information in our data processing, we fused the images from average projection and correlation map extracting the temporal information of active neurons, and the integrated information was expressed as two-dimensional (2D) images. To achieve a generalized neuron segmentation, we conducted a hybrid learning strategy by training our model with imaging data from different labs, including multiscale data with different Ca2+ indicators. The results showed that our approach achieved promising segmentation performance across different imaging scales and Ca2+ indicators, even including the challenging data of large field-of-view mesoscopic images. By comparing state-of-the-art neuron segmentation methods for two-photon Ca2+ imaging data, we showed that our approach achieved the highest accuracy with a publicly available dataset. Thus, NeuroSeg-II enables good segmentation accuracy and a convenient training and testing process.
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The ability to form predictions based on recent sensory experience is essential for behavioral adaptation to our ever-changing environment. Predictive encoding represented by neuronal activity has been observed in sensory cortex, but how this neuronal activity is transformed into anticipatory motor behavior remains unclear. Fiber photometry to investigate a corticostriatal projection from the auditory cortex to the posterior striatum during an auditory paradigm in mice, and pharmacological experiments in a task that induces a temporal expectation of upcoming sensory stimuli. We find that the auditory corticostriatal projection relays both sound-evoked stimulus information as well as predictive signals in relation to stimulus timing following rhythmic auditory stimulation. Pharmacological experiments suggest that this projection is required for the initiation of both sound-evoked and anticipatory licking behavior in an auditory associative-learning behavioral task, but not for the general recognition of presented auditory stimuli. This auditory corticostriatal projection carries predictive signals, and the posterior striatum is critical to the anticipatory stimulus-driven motor behavior.
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Corteza Auditiva , Sonido , Animales , Ratones , Estimulación Acústica , Neostriado , CogniciónRESUMEN
Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations in vivo. Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca2+ imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca2+ imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.
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Significance: The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. Aim: We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400 kHz and a maximum frame rate of 10,000 frames per second (fps) at 250 × 40 pixels . For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. Approach: With this combinatorial device that supports both large-view navigation and small-view high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). Results: We measured dendritic calcium propagation velocity ( 80 / 62.5 - 116.7 µ m / ms ) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow. Conclusions: This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo.
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The prevalence and disease burden of urolithiasis has increased substantially worldwide in the last decade, and intraluminal holmium laser lithotripsy has become the primary treatment method. However, inappropriate laser energy settings increase the risk of perioperative complications, largely due to the lack of intraoperative information on the stone composition, which determines the stone melting point. To address this issue, we developed a fiber-based fluorescence spectrometry method that detects and classifies the autofluorescence spectral fingerprints of urinary stones into three categories: calcium oxalate, uric acid, and struvite. By applying the support vector machine (SVM), the prediction accuracy achieved 90.28 % and 96.70% for classifying calcium stones versus non-calcium stones and uric acid versus struvite, respectively. High accuracy and specificity were achieved for a wide range of working distances and angles between the fiber tip and stone surface in an emulated intraoperative ambient. Our work establishes the methodological basis for engineering a clinical device that achieves real-time, in situ classification of urinary stones for optimizing the laser ablation parameters and reducing perioperative complications in lithotripsy.