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Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.
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Encéfalo/fisiologia , Dendritos/fisiologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/fisiologia , Sinapses/fisiologia , Algoritmos , Animais , Cálcio/química , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Radiocirurgia , Ácido gama-AminobutíricoRESUMO
The ability to not only control but also maintain the well-defined size of nanoclusters is key to a scientific understanding as well as their practical application. Here, we report a synthetic protocol to prepare and stabilize nanoclusters of different metals and even metal salts. The approach builds on a Pickering stabilization effect inside a microemulsion system. We prove that the emulsion interface plays a critical role in the formation of nanoclusters, which are encapsulated in situ into a silica matrix. The resulting nanocapsule is characterized by a central cavity and a porous shell composed of a matrix of both silica and nanoclusters. This structure endows the nanoclusters simultaneously with high thermal stability, good biocompatibility, and excellent photostability, making them well suited for fundamental studies and practical applications ranging from materials chemistry, catalysis, and optics to bioimaging.
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Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.
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Corpo Caloso/fisiologia , Neurônios/fisiologia , Estimulação Luminosa/métodos , Córtex Visual Primário/fisiologia , Vias Visuais/fisiologia , Animais , Corpo Caloso/química , Corpo Caloso/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/química , Córtex Visual Primário/química , Córtex Visual Primário/citologia , Vias Visuais/química , Vias Visuais/citologiaRESUMO
Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.
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Corantes/química , Processamento de Imagem Assistida por Computador/métodos , Coloração e Rotulagem/métodos , Animais , Encéfalo/anatomia & histologia , Linhagem Celular , Drosophila , Larva/citologia , Camundongos , Microscopia de Fluorescência , Processos FotoquímicosRESUMO
Volumetric imaging tools that are simple to adopt, flexible, and robust are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here, we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module that allowed for the generation of Bessel foci of varying numerical apertures and axial lengths, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.
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Encéfalo/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/instrumentação , Imageamento Tridimensional/instrumentação , Luz , Microscopia de Fluorescência por Excitação Multifotônica , Animais , Simulação por Computador , Radiação Eletromagnética , Desenho de Equipamento , Análise de Fourier , Camundongos , Micromanipulação , MicroscopiaRESUMO
The crystallinity of cadmium titanate (CdTiO3) was greatly improved when synthesized under mild reaction conditions, in the presence of chloride. The highly crystalline CdTiO3 showed much enhanced photodegradation of methyl orange (MO) under simulated sunlight. CdTiO3 was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), N2 adsorption/desorption, photoluminescence (PL), and UV/vis spectrometry. The enhanced photodegradation was attributed to the better charge separation owing to its higher crystallinity.
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Virtually structured detection (VSD) has been demonstrated to break the diffraction limit in scanning laser microscopy (SLM). VSD provides an easy, low-cost, and phase-artifact-free strategy to achieve super-resolution imaging. However, practical application of this method is challenging due to a limited image acquisition speed. We report here the combination of VSD and line-scanning microscopy (LSM) to improve the image acquisition speed. A motorized dove prism was used to achieve automatic control of four-angle (i.e., 0°, 45°, 90°, and 135°) scanning, thus ensuring isotropic resolution improvement. Both an optical resolution target and a living frog eyecup were used to verify resolution enhancement.
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Microscopia Confocal/métodos , Animais , Anuros , Processamento de Imagem Assistida por Computador , Retina/citologia , Razão Sinal-Ruído , Fatores de TempoRESUMO
Dynamic near infrared microscopy has revealed transient retinal phototropism (TRP) correlated with oblique light stimulation. Here, by developing a hybrid confocal microscopy and optical coherence tomography (OCT), we tested sub-cellular source of the TRP in living frog retina. Dynamic confocal microscopy and OCT consistently revealed photoreceptor outer segments as the anatomic source of the TRP. Further investigation of the TRP can provide insights in better understanding of Stiles-Crawford effect (SCE) on rod and cone systems, and may also promise an intrinsic biomarker for early detection of eye diseases that can produce photoreceptor dysfunction.
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Luz , Segmento Externo das Células Fotorreceptoras da Retina/efeitos da radiação , Tomografia de Coerência Óptica , Animais , Anuros , Estudos de ViabilidadeRESUMO
Functional mesoporous carbon nanomaterials with large pores and small particle sizes have broad accessibility, but remain challenging to achieve. This study proposed a dual-template synergistic assembly strategy to facilely synthesize extra-small nitrogen-doped mesoporous carbon nanospheres with large pores in a low-cost manner. Directed by the synergistic effect of the combination of surfactants, sodium oleate (anionic surfactant) and triblock copolymer-P123 (nonionic surfactant) were selected as templates to construct nanomicelles (nanoemulsions), which were co-assembled with melamine-based oligomers to form composite nanomicelles, thus obtaining nitrogen-doped mesoporous polymer nanospheres (NMePS) and then nitrogen-doped mesoporous carbon nanospheres (NMeCS). Based on Schiff base chemistry, the melamine-based oligomers with self-assembly capability were synthesized as precursors, which is different from the conventional synthetic route of melamine-formaldehyde resin. The key parameters involved in the route were investigated comprehensively and correlated with the characterization results. Furthermore, the 50 nm-scale particle size and the large mesoporous size of 5.5 nm of NMeCS can facilitate effective mass transport, coupled with their high nitrogen content (15.7 wt%), contributing to their excellent performance in lithium-ion batteries.
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Phase change materials (PCMs) possess the potential to regulate temperature by utilizing their thermal properties to absorb and release heat. Nevertheless, the application of PCMs in thermal management is constrained by issues such as liquid leakage and limited flexibility. In this study, we propose a novel approach to address these challenges by incorporating a pore structure within nanofibers to confine the crystallization of phase change molecules, thereby enhancing the flexibility of the composite material. Additionally, inspired by the adaptive mechanisms observed in plants, we have developed a form stable PCM based on polyether, which effectively mitigates the issue of liquid leakage at higher temperatures. Despite being a solid-liquid PCM at its core, this material exhibits molecular-scale flow and macroscopic shape stability as a result of intermolecular forces. The composite film material possesses remarkable flexibility, efficient thermal management capabilities, adjustable phase transition temperature, and the ability to undergo repeated processing and utilization. Consequently, it holds promising potential for applications in personal thermal energy management.
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This study was designed to test the feasibility of en face mapping of the transient intrinsic optical signal (IOS) response at photoreceptor outer segments and to assess the effect of spatial resolution on functional IOS imaging of retinal photoreceptors. A line-scan optical coherence tomography (LS-OCT) was constructed to achieve depth-resolved functional IOS imaging of living frog eyecups. Rapid en face OCT revealed transient IOS almost immediately (<3 ms) after the onset of visible light flashes at photoreceptor outer segments. Quantitative analysis indicated that the IOS kinetics may reflect dynamics of G-protein binding and releasing in early phases of visual transduction, and high resolution is essential to differentiate positive and negative IOS changes in adjacent locations.
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Interpretação de Imagem Assistida por Computador/métodos , Estimulação Luminosa/métodos , Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Tomografia de Coerência Óptica/métodos , Animais , Luz , Células Fotorreceptoras de Vertebrados/ultraestrutura , RanidaeRESUMO
Orientation columns exist in the primary visual cortex (V1) of cat and primates but not mouse. Intriguingly, some recent studies reported the presence of orientation and direction columns in the mouse superficial superior colliculus (sSC), while others reported a lack of columnar organization therein. Using in vivo calcium imaging of sSC in the awake mouse brain, we found that the presence of columns is highly stimulus dependent. Specifically, we observed orientation and direction columns formed by sSC neurons retinotopically mapped to the edge of grating stimuli. For both excitatory and inhibitory neurons in sSC, orientation selectivity can be induced by the edge with their preferred orientation perpendicular to the edge orientation. Furthermore, we found that this edge-induced orientation selectivity is associated with saliency encoding. These findings indicate that the tuning properties of sSC neurons are not fixed by circuit architecture but rather dependent on the spatiotemporal properties of the stimulus.
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Colículos Superiores , Córtex Visual , Animais , Colículos Superiores/fisiologia , Córtex Visual/fisiologia , Estimulação Luminosa/métodos , Neurônios/fisiologia , CálcioRESUMO
Building shape-stable phase change materials (PCMs) are crucial for their practical applications. Particularly, it is vital to utilize renewable/recyclable biomass media as the support material of form-stable PCMs. In this review article, we summarized the recent developments for building form-stable PCMs consisting of wood as a supporting material, either carbonized wood or wood composites. Moreover, the electrothermal conversion and photothermal conversion of form-stable PCMs based on carbonized wood are also demonstrated. In addition, the current technical problems and future research developments of wood-based PCMs are discussed, especially the leakage problem of PCMs during the phase change transition process. All of this information will be helpful for the in-depth understanding and development of new PCMs suitable for wide application perspectives.
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Functional measurement is important for retinal study and disease diagnosis. Transient intrinsic optical signal (IOS) response, tightly correlated with functional stimulation, has been previously detected in normal retinas. In this paper, comparative IOS imaging of wild-type (WT) and rod-degenerated mutant mouse retinas is reported. Both 2-month and 1-year-old mice were measured. In 2-month-old mutant mice, time course and peak value of the stimulus-evoked IOS were significantly delayed (relative to stimulus onset) and reduced, respectively, compared to age matched WT mice. In 1-year-old mutant mice, stimulus-evoked IOS was totally absent. However, enhanced spontaneous IOS responses, which might reflect inner neural remodeling in diseased retina, were observed in both 2-month and 1-year-old mutant retinas. Our experiments demonstrate the potential of using IOS imaging for noninvasive and high resolution identification of disease-associated retinal dysfunctions. Moreover, high spatiotemporal resolution IOS imaging may also lead to advanced understanding of disease-associated neural remodeling in the retina.
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Potenciais Evocados Visuais , Doenças Retinianas/diagnóstico , Doenças Retinianas/fisiopatologia , Neurônios Retinianos , Retinoscópios , Animais , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Adaptive optics reflectance-based retinal imaging has proved a valuable tool for the noninvasive visualization of cells in the living human retina. Many subcellular features that remain at or below the resolution limit of current in vivo techniques may be more easily visualized with the same modalities in an ex vivo setting. While most microscopy techniques provide significantly higher resolution, enabling the visualization of fine cellular detail in ex vivo retinal samples, they do not replicate the reflectance-based imaging modalities of in vivo retinal imaging. Here, we introduce a strategy for imaging ex vivo samples using the same imaging modalities as those used for in vivo retinal imaging, but with increased resolution. We also demonstrate the ability of this approach to perform protein-specific fluorescence imaging and reflectance imaging simultaneously, enabling the visualization of nearly transparent layers of the retina and the classification of cone photoreceptor types.
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Choroideremia is an X-linked, blinding retinal degeneration with progressive loss of photoreceptors, retinal pigment epithelial (RPE) cells, and choriocapillaris. To study the extent to which these layers are disrupted in affected males and female carriers, we performed multimodal adaptive optics imaging to better visualize the in vivo pathogenesis of choroideremia in the living human eye. We demonstrate the presence of subclinical, widespread enlarged RPE cells present in all subjects imaged. In the fovea, the last area to be affected in choroideremia, we found greater disruption to the RPE than to either the photoreceptor or choriocapillaris layers. The unexpected finding of patches of photoreceptors that were fluorescently-labeled, but structurally and functionally normal, suggests that the RPE blood barrier function may be altered in choroideremia. Finally, we introduce a strategy for detecting enlarged cells using conventional ophthalmic imaging instrumentation. These findings establish that there is subclinical polymegathism of RPE cells in choroideremia.
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Coroideremia , Degeneração Retiniana , Corioide/diagnóstico por imagem , Coroideremia/genética , Coroideremia/patologia , Feminino , Humanos , Masculino , Óptica e Fotônica , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana/patologiaRESUMO
Simultaneous monitoring of many functioning ß-cells is essential for understanding ß-cell dysfunction as an early event in the progression to diabetes. Intrinsic optical signal (IOS) imaging has been shown to allow high resolution detection of stimulus-evoked physiological responses in the retina and other neural tissues. In this paper, we demonstrate the feasibility of using IOS imaging for functional examination of insulin secreting INS-1 cells, a popular model for investigating diabetes associated ß-cell dysfunction. Our experiments indicate that IOS imaging permits simultaneous monitoring of glucose-stimulated physiological responses in multiple cells with high spatial (sub-cellular) and temporal (sub-second) resolution. Rapid IOS image sequences revealed transient optical responses that had time courses tightly correlated with the glucose stimulation.
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Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Animais , Linhagem Celular , Diagnóstico por Imagem , Glucose/farmacologia , Raios Infravermelhos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Fenômenos Ópticos , Ratos , Transdução de SinaisRESUMO
Linear polarization intrinsic optical signal (LP-IOS) measurement can provide sensitive detection of neural activities in stimulus-activated neural tissues. However, the LP-IOS magnitude and signal-to-noise ratio (SNR) are highly correlated with the nerve orientation relative to the polarization plane of the incident light. Because of the complexity of orientation dependency, LP-IOS optimization and outcome interpretation are time consuming and complicated. In this study, we demonstrate the feasibility of circular polarization intrinsic optical signal (CP-IOS) measurement. Our theoretical modeling and experimental investigation indicate that CP-IOS magnitude and SNR are independent from the nerve orientation. Therefore, CP-IOS promises a practical method for polarization IOS imaging of complex neural systems.
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Modelos Biológicos , Fenômenos Fisiológicos do Sistema Nervoso , Fenômenos Ópticos , Axônios/metabolismo , Fenômenos Eletrofisiológicos , Estudos de Viabilidade , Sistema Nervoso/citologiaRESUMO
Using freshly isolated animal retinas, we have conducted a series of experiments to test fast intrinsic optical signals (IOSs) that have time courses comparable to electrophysiological kinetics. In this Letter, we demonstrate the feasibility of in vivo imaging of fast IOSs in intact frogs. A rapid line-scan confocal ophthalmoscope was constructed to achieve high-speed IOS recording. By rejecting out-of-focus background light, the line-scan confocal imager provided the resolution to differentiate individual photoreceptors in vivo. Rapid confocal imaging disclosed robust IOSs with time courses comparable to retinal electroretinogram kinetics. High-resolution IOS images revealed both positive (increasing) and negative (decreasing) light responses, with subcellular complexity.
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Microscopia Confocal/métodos , Oftalmoscopia/métodos , Retina/fisiologia , Animais , Fenômenos Eletrofisiológicos , Microscopia Confocal/instrumentação , Oftalmoscópios , Fenômenos Ópticos , Estimulação Luminosa , Células Fotorreceptoras de Vertebrados/fisiologia , Rana pipiensRESUMO
Quantitative retinal imaging is essential for advanced study and clinical management of eye diseases. However, spatial resolution of retinal imaging has been limited due to available numerical aperture and optical aberration of the ocular optics. Structured illumination microscopy has been established to break the diffraction-limit resolution in conventional light microscopy. However, practical implementation of structured illumination microscopy for in vivo ophthalmoscopy of the retina is challenging due to inevitable eye movements that can produce phase artifacts. Recently, we have demonstrated the feasibility of using virtually structured detection as one alternative to structured illumination microscopy for super-resolution imaging. By providing the flexibility of digital compensation of eye movements, the virtually structured detection provides a feasible, phase-artifact-free strategy to achieve super-resolution ophthalmoscopy. In this article, we summarize the technical rationale of virtually structured detection, and its implementations for super-resolution imaging of freshly isolated retinas, intact animals, and awake human subjects.