RESUMO
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.
Assuntos
Encéfalo , Aprendizado Profundo , Imageamento Tridimensional , Humanos , Imageamento Tridimensional/métodos , Encéfalo/diagnóstico por imagem , Algoritmos , Neurônios/citologia , Microscopia de Fluorescência/métodosRESUMO
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.
Assuntos
Encéfalo , Peróxido de Hidrogênio , Humanos , Microscopia de Fluorescência/métodos , Encéfalo/diagnóstico por imagem , Neurônios , Neuroimagem/métodos , Imageamento Tridimensional , Imagem Óptica/métodosRESUMO
T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
Assuntos
Miócitos Cardíacos , Optogenética , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Membrana Celular , Potenciais da Membrana , Potenciais de Ação/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.
RESUMO
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.
Assuntos
Área de Broca , Córtex Cerebral , Humanos , Encéfalo/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Mapeamento EncefálicoRESUMO
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.
Assuntos
Peróxido de Hidrogênio , Imageamento Tridimensional , Adulto , Idoso , Encéfalo/diagnóstico por imagem , Imunofluorescência , Humanos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodosRESUMO
Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.
Assuntos
Adenilil Ciclases , Canal de Liberação de Cálcio do Receptor de Rianodina , Adenilil Ciclases/metabolismo , Adrenérgicos , Cálcio/metabolismo , Sinalização do Cálcio , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Cover-all mapping of the distribution of neurons in the human brain would have a significant impact on the deep understanding of brain function. Therefore, complete knowledge of the structural organization of different human brain regions at the cellular level would allow understanding their role in the functions of specific neural networks. Recent advances in tissue clearing techniques have allowed important advances towards this goal. These methods use specific chemicals capable of dissolving lipids, making the tissue completely transparent by homogenizing the refractive index. However, labeling and clearing human brain samples is still challenging. Here, we present an approach to perform the cellular mapping of the human cerebral cortex coupling immunostaining with SWITCH/TDE clearing and confocal microscopy. A specific evaluation of the contributions of the autofluorescence signals generated from the tissue fixation is provided as well as an assessment of lipofuscin pigments interference. Our evaluation demonstrates the possibility of obtaining an efficient clearing and labeling process of parts of adult human brain slices, making it an excellent method for morphological classification and antibody validation of neuronal and non-neuronal markers.
Assuntos
Encéfalo , Neurônios , Córtex Cerebral , Humanos , Imageamento Tridimensional , Microscopia ConfocalRESUMO
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.
RESUMO
RATIONALE: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.
Assuntos
Acoplamento Excitação-Contração , Frequência Cardíaca , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Potenciais de Ação , Animais , Difusão , Tomografia com Microscopia Eletrônica , Feminino , Recuperação de Fluorescência Após Fotodegradação , Miócitos Cardíacos/ultraestrutura , Coelhos , Sarcolema/ultraestruturaRESUMO
Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms.
RESUMO
Disopyramide is effective and safe in patients with obstructive hypertrophic cardiomyopathy. However, its cellular and molecular mechanisms of action are unknown. We tested disopyramide in cardiomyocytes from the septum of surgical myectomy patients: disopyramide inhibits multiple ion channels, leading to lower Ca transients and force, and shortens action potentials, thus reducing cellular arrhythmias. The electrophysiological profile of disopyramide explains the efficient reduction of outflow gradients but also the limited prolongation of the QT interval and the absence of arrhythmic side effects observed in 39 disopyramide-treated patients. In conclusion, our results support the idea that disopyramide is safe for outpatient use in obstructive patients.
RESUMO
Asynchronous Ca2+ release promotes non-homogeneous myofilament activation, leading to mechanical dysfunction, as well as initiation of propagated calcium waves and arrhythmias. Recent advances in microscopy techniques have allowed for optical recordings of local Ca2+ fluxes and action potentials from multiple sub-cellular domains within cardiac cells with unprecedented spatial and temporal resolution. Since then, sub-cellular local information of the spatio-temporal relationship between Ca2+ release and action potential propagation have been unlocked, providing novel mechanistic insights in cardiac excitation-contraction coupling (ECC). Here, we review the promising perspectives arouse from repeatedly probing Ca2+ release at the same sub-cellular location while simultaneously probing multiple locations at the same time within a single cardiac cell. We also compare the results obtained in three different rodent models of cardiac diseases, highlighting disease-specific mechanisms. Slower local Ca2+ release has been observed in regions with defective action potential conduction in diseased cardiac cells. Moreover, significant increment of Ca2+ variability (both in time and in space) has been found in diseased cardiac cells but does not directly correlate with local electrical defects nor with the degree of structural aberrations of the cellular membrane system, suggesting a role for other players of the ECC machinery. We finally explore exciting opportunities provided by the technology for studying different cardiomyocyte populations, as well as for dissecting the mechanisms responsible for subcellular spatio-temporal variability of Ca2+ release.
RESUMO
Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling. Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human. Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections. Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial "super-hub" Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.
RESUMO
Current rescue therapies for life-threatening arrhythmias ignore the pathological electro-anatomical substrate and base their efficacy on a generalized electrical discharge. Here, we developed an all-optical platform to examine less invasive defibrillation strategies. An ultrafast wide-field macroscope was developed to optically map action potential propagation with a red-shifted voltage sensitive dye in whole mouse hearts. The macroscope was implemented with a random-access scanning head capable of drawing arbitrarily-chosen stimulation patterns with sub-millisecond temporal resolution allowing precise epicardial activation of Channelrhodopsin2 (ChR2). We employed this optical system in the setting of ventricular tachycardia to optimize mechanistic, multi-barrier cardioversion/defibrillation patterns. Multiple regions of conduction block were created with a very high cardioversion efficiency but with lower energy requirements as compared to whole ventricle interventions to interrupt arrhythmias. This work demonstrates that defibrillation energies can be substantially reduced by applying discrete stimulation patterns and promotes the progress of current anti-arrhythmic strategies.