Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

University of Florence

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.

Optogenetics design of mechanistically-based stimulation patterns for cardiac defibrillation.

 Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca 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) Ca 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.  To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca signaling proteins across species from mouse to human.  We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye  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 Ca channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca current was similar in human and mouse AMs, the intracellular Ca transient showed quantitative differences. Importantly, local intracellular Ca 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 Ca release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections.  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" Ca signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.

Axial Tubule Junctions Activate Atrial Ca Release Across Species.

Asynchronous Ca 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 Ca 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 Ca 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 Ca 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 Ca release has been observed in regions with defective action potential conduction in diseased cardiac cells. Moreover, significant increment of Ca 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 Ca release.

Interplay Between Sub-Cellular Alterations of Calcium Release and T-Tubular Defects in Cardiac Diseases.

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.

A Software Architecture to Mimic a Ventricular Tachycardia in Intact Murine Hearts by Means of an All-Optical Platform.

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.

Electrophysiological and Contractile Effects of Disopyramide in Patients With Obstructive Hypertrophic Cardiomyopathy: A Translational Study.

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.

Exploring the human cerebral cortex using confocal microscopy.

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.

Comparison of Different Tissue Clearing Methods for Three-Dimensional Reconstruction of Human Brain Cellular Anatomy Using Advanced Imaging Techniques.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca-induced Ca release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following β-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 Ca 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 β-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 Ca 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 Ca current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca-induced Ca release, whereas β-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

A junctional cAMP compartment regulates rapid Ca signaling in atrial myocytes.

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-HO-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.

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy.

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.

A cellular resolution atlas of Broca's area.

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  MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in  MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere  scans at 120 m, 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.

Marina Scardigli

European Laboratory for Non-linear Spectroscopy (LENS),
Division of Physiology,
Department of Experimental and Clinical Medicine,
Division of Physiology, Department of Experimental and Clinical Medicine

University of Florence

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Marina Scardigli

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}European Laboratory for Non-linear Spectroscopy (LENS),Division of Physiology,Department of Experimental and Clinical Medicine,Division of Physiology, Department of Experimental and Clinical Medicine

University of Florence

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

European Laboratory for Non-linear Spectroscopy (LENS),
Division of Physiology,
Department of Experimental and Clinical Medicine,
Division of Physiology, Department of Experimental and Clinical Medicine