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Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.
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Colestanos , Animais , Camundongos , Colestanos/farmacologia , Espermina/farmacologia , Microscopia de Fluorescência/métodos , ColesterolRESUMO
The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology.
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Imageamento Tridimensional , Hematoxilina , Amarelo de Eosina-(YS) , Microscopia de Fluorescência/métodos , Coloração e Rotulagem , Imageamento Tridimensional/métodos , Microscopia ConfocalRESUMO
Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.
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Encéfalo , Peróxido de Hidrogênio , Humanos , Microscopia de Fluorescência/métodos , Encéfalo/diagnóstico por imagem , Neurônios , Neuroimagem/métodos , Imageamento Tridimensional , Imagem Óptica/métodosRESUMO
3D reconstruction of human brain volumes at high resolution is now possible thanks to advancements in tissue clearing methods and fluorescence microscopy techniques. Analyzing the massive data produced with these approaches requires automatic methods able to perform fast and accurate cell counting and localization. Recent advances in deep learning have enabled the development of various tools for cell segmentation. However, accurate quantification of neurons in the human brain presents specific challenges, such as high pixel intensity variability, autofluorescence, non-specific fluorescence and very large size of data. In this paper, we provide a thorough empirical evaluation of three techniques based on deep learning (StarDist, CellPose and BCFind-v2, an updated version of BCFind) using a recently introduced three-dimensional stereological design as a reference for large-scale insights. As a representative problem in human brain analysis, we focus on a 4 -cm 3 portion of the Broca's area. We aim at helping users in selecting appropriate techniques depending on their research objectives. To this end, we compare methods along various dimensions of analysis, including correctness of the predicted density and localization, computational efficiency, and human annotation effort. Our results suggest that deep learning approaches are very effective, have a high throughput providing each cell 3D location, and obtain results comparable to the estimates of the adopted stereological design.
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Encéfalo , Aprendizado Profundo , Imageamento Tridimensional , Humanos , Imageamento Tridimensional/métodos , Encéfalo/diagnóstico por imagem , Algoritmos , Neurônios/citologia , Microscopia de Fluorescência/métodosRESUMO
Sensory information must be integrated across a distributed brain network for stimulus processing and perception. Recent studies have revealed specific spatiotemporal patterns of cortical activation for the early and late components of sensory-evoked responses, which are associated with stimulus features and perception, respectively. Here, we investigated how the brain state influences the sensory-evoked activation across the mouse cortex. We utilized isoflurane to modulate the brain state and conducted wide-field calcium imaging of Thy1-GCaMP6f mice to monitor distributed activation evoked by multi-whisker stimulation. Our findings reveal that the level of anesthesia strongly shapes the spatiotemporal features and the functional connectivity of the sensory-activated network. As anesthesia levels decrease, we observe increasingly complex responses, accompanied by the emergence of the late component within the sensory-evoked response. The persistence of the late component under anesthesia raises new questions regarding the potential existence of perception during unconscious states.
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Fluorescence microscopy can be exploited for evaluating the brain's fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.
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Algoritmos , Encéfalo , Encéfalo/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Imagem de Difusão por Ressonância Magnética/métodos , Microscopia de FluorescênciaRESUMO
Fear responses are functionally adaptive behaviors that are strengthened as memories. Indeed, detailed knowledge of the neural circuitry modulating fear memory could be the turning point for the comprehension of this emotion and its pathological states. A comprehensive understanding of the circuits mediating memory encoding, consolidation, and retrieval presents the fundamental technological challenge of analyzing activity in the entire brain with single-neuron resolution. In this context, we develop the brain-wide neuron quantification toolkit (BRANT) for mapping whole-brain neuronal activation at micron-scale resolution, combining tissue clearing, high-resolution light-sheet microscopy, and automated image analysis. The robustness and scalability of this method allow us to quantify the evolution of activity patterns across multiple phases of memory in mice. This approach highlights a strong sexual dimorphism in recruited circuits, which has no counterpart in the behavior. The methodology presented here paves the way for a comprehensive characterization of the evolution of fear memory.
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Encéfalo , Caracteres Sexuais , Camundongos , Animais , Encéfalo/fisiologia , Medo/fisiologia , Neurônios/fisiologiaRESUMO
Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Leveraging recent advancements in ultra-high resolution ex vivo MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in ex vivo MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere ex vivo scans at 120 µm, we propose a multi-resolution U-Nets framework (MUS) that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation, while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.
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Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)-referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca's area in a human postmortem specimen, within a whole-brain reference space atlas.
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Área de Broca , Córtex Cerebral , Humanos , Encéfalo/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Mapeamento EncefálicoRESUMO
SIGNIFICANCE: Most radiative transport problems in turbid media are typically associated with mm or cm scales, leading to typical time scales in the range of hundreds of ps or more. In certain cases, however, much thinner layers can also be relevant, which can dramatically alter the overall transport properties of a scattering medium. Studying scattering in these thin layers requires ultrafast detection techniques and adaptations to the common Monte Carlo (MC) approach. AIM: We aim to discuss a few relevant aspects for the simulation of light transport in thin scattering membranes, and compare the obtained numerical results with experimental measurements based on an all-optical gating technique. APPROACH: A thin membrane with controlled scattering properties based on polymer-dispersed TiO2 nanoparticles is fabricated for experimental validation. Transmittance measurements are compared against a custom open-source MC implementation including specific pulse profiles for tightly focused femtosecond laser pulses. RESULTS: Experimental transmittance data of ultrafast pulses through a thin scattering sample are compared with MC simulations in the spatiotemporal domain to retrieve its scattering properties. The results show good agreement also at short distances and time scales. CONCLUSIONS: When simulating light transport in scattering membranes with thicknesses in the orders of tens of micrometer, care has to be taken when describing the temporal, spatial, and divergence profiles of the source term, as well as the possible truncation of step length distributions, which could be introduced by simple strategies for the generation of random exponentially distributed variables.
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Diagnóstico por Imagem , Nanopartículas , Simulação por Computador , Frequência Cardíaca , Método de Monte CarloRESUMO
Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).
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The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH-H2O2-antigen Retrieval-TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.
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Peróxido de Hidrogênio , Imageamento Tridimensional , Adulto , Idoso , Encéfalo/diagnóstico por imagem , Imunofluorescência , Humanos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodosRESUMO
The Brain Imaging Data Structure (BIDS) is a specification for organizing, sharing, and archiving neuroimaging data and metadata in a reusable way. First developed for magnetic resonance imaging (MRI) datasets, the community-led specification evolved rapidly to include other modalities such as magnetoencephalography, positron emission tomography, and quantitative MRI (qMRI). In this work, we present an extension to BIDS for microscopy imaging data, along with example datasets. Microscopy-BIDS supports common imaging methods, including 2D/3D, ex/in vivo, micro-CT, and optical and electron microscopy. Microscopy-BIDS also includes comprehensible metadata definitions for hardware, image acquisition, and sample properties. This extension will facilitate future harmonization efforts in the context of multi-modal, multi-scale imaging such as the characterization of tissue microstructure with qMRI.
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The combination of tissue clearing techniques with advanced optical microscopy facilitates the achievement of three-dimensional (3D) reconstruction of macroscopic specimens at high resolution. Whole mouse organs or even bodies have been analyzed, while the reconstruction of the human nervous system remains a challenge. Although several tissue protocols have been proposed, the high autofluorescence and variable post-mortem conditions of human specimens negatively affect the quality of the images in terms of achievable transparency and staining contrast. Moreover, homogeneous staining of high-density epitopes, such as neuronal nuclear antigen (NeuN), creates an additional challenge. Here, we evaluated different tissue transformation approaches to find the best solution to uniformly clear and label all neurons in the human cerebral cortex using anti-NeuN antibodies in combination with confocal and light-sheet fluorescence microscopy (LSFM). Finally, we performed mesoscopic high-resolution 3D reconstruction of the successfully clarified and stained samples with LSFM.
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Analyzing the structure of neuronal fibers with single axon resolution in large volumes is a challenge in connectomics. Different technologies try to address this goal; however, they are limited either by the ineffective labeling of the fibers or in the achievable resolution. The possibility of discriminating between different adjacent myelinated axons gives the opportunity of providing more information about the fiber composition and architecture within a specific area. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a tissue preparation method to perform label-free fluorescence imaging of myelinated fibers that is user friendly and easy to handle. We exploit the high axial and radial resolution of two-photon fluorescence microscopy (TPFM) optical sectioning to decipher the mixture of various fiber orientations within the sample of interest. We demonstrate its broad applicability by performing mesoscopic reconstruction at a sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in control and Reeler mouse's hippocampal sections. Our study provides a novel method for 3D label-free imaging of nerve fibers in fixed samples at high resolution, below micrometer level, that overcomes the limitation related to the myelinated axons exogenous labeling, improving the possibility of analyzing brain connectivity.
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Encéfalo , Fluorescência , Fibras Nervosas Mielinizadas , Animais , Humanos , Camundongos , RatosRESUMO
Although neuronal density analysis on human brain slices is available from stereological studies, data on the spatial distribution of neurons in 3D are still missing. Since the neuronal organization is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than relying on sparse sampling methods. To achieve this goal, we implement a new tissue transformation protocol to clear and label human brain tissues and we exploit the high-resolution optical sectioning of two-photon fluorescence microscopy to perform 3D mesoscopic reconstruction. We perform neuronal mapping of 100mm3 human brain samples and evaluate the volume and density distribution of neurons from various areas of the cortex originating from different subjects (young, adult, and elderly, both healthy and pathological). The quantitative evaluation of the density in combination with the mean volume of the thousands of neurons identified within the specimens, allow us to determine the layer-specific organization of the cerebral architecture.
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Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution and, in combination with tissue clearing methods, is often employed to reconstruct the cyto-architecture over the intact mouse brain. Inherently to LSM, however, residual opaque objects, always present to some extent even in extremely well optically cleared samples, cause stripe artifacts, which, in the best case, severely affect image homogeneity and, in the worst case, completely obscure features of interest. Here, demonstrating two example applications in intact optically cleared mouse brains, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data for the brain-wide morphology of neuronal and vascular networks. We found that a third of the imaged volume of the brain was affected by strong striated image intensity inhomogeneity and, furthermore, a significant amount of information content lost with Gaussian illumination was accessible when interrogated with Bessel beams. In conclusion, Bessel beams produce high-fidelity structural data of improved image homogeneity and might significantly relax demands placed on the automated tools to count, trace, or segment fluorescent features of interest.
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Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Microscopia Confocal , Neurônios/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal/métodos , Distribuição Normal , Parvalbuminas/genética , Parvalbuminas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/patologiaRESUMO
Tremendous enhancement of light-matter interaction in plasmonic-dielectric hybrid devices allows for non-linearities at the level of single emitters and few photons, such as single photon transistors. However, constructing integrated components for such devices is technologically extremely challenging. We tackle this task by lithographically fabricating an on-chip plasmonic waveguide-structure connected to far-field in- and out-coupling ports via low-loss dielectric waveguides. We precisely describe our lithographic approach and characterize the fabricated integrated chip. We find excellent agreement with rigorous numerical simulations. Based on these findings we perform a numerical optimization and calculate concrete numbers for a plasmonic single-photon transistor.