High-speed imaging with light-sheet fluorescence microscopy poses several challenges throughout the whole pipeline, from data acquisition in the lab to image stitching and post-processing. Here we present our custom hardware and software solutions that allow us to map large biological samples at the cellular level, e.g. large portions of human brain cortex. Our custom optical setup—a dual-view, inverted, light-sheet microscope—is capable of simultaneous two-color acquisition at a data rate of 1 GB/s. Our open source tools include the instrument’s data acquisition and control software and also cover volumetric image stitching and post-processing.
3D reconstruction of the human brain at high resolution is one of the most important challenges of neuroscience. We present a new clearing method named SHORT that in combination with an advanced double-view light-sheet fluorescence microscope and an automated machine-learning based images analysis allow to perform volumetric study of the human brain. We applied our methodology to a Broca’s area block of 4 x 4 x 2 cm3, demonstrating the possibility of obtaining a fast 3D reconstruction of the human brain at high-resolution, paving the way to the possibility of finally mapping a comprehensive atlas of the human brain.
This conference presentation, “3D molecular phenotyping of the human brain Broca’s area using light-sheet fluorescence microscopy” was prepared for the Biomedical Spectroscopy, Microscopy, and Imaging II conference at SPIE Photonics Europe 2022.
Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics’ agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, in this article we review an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion article, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed and provide an outlook for the future directions.
The signal of optical coherence tomography (OCT) decays exponentially in depth due to tissue scattering, resulting in indistinct tissue features in three-dimension. Moreover, due to limited light penetration depth, extensive volumetric investigation is usually constrained for large-scale biological samples. By integrating serial sectioning technology with block-face imaging, we establish a volumetric OCT acquisition and reconstruction pipeline that incorporates depth-resolved attenuation coefficient estimation, volumetric stitching and filtering, and feature enhancement visualization. We demonstrate this pipeline on ex vivo human brain volumes of several cubic centimeters with 5 um isotropic resolution.
Significance: The optical properties of biological samples provide information about the structural characteristics of the tissue and any changes arising from pathological conditions. Optical coherence tomography (OCT) has proven to be capable of extracting tissue’s optical properties using a model that combines the exponential decay due to tissue scattering and the axial point spread function that arises from the confocal nature of the detection system, particularly for higher numerical aperture (NA) measurements. A weakness in estimating the optical properties is the inter-parameter cross-talk between tissue scattering and the confocal parameters defined by the Rayleigh range and the focus depth.
Aim: In this study, we develop a systematic method to improve the characterization of optical properties with high-NA OCT.
Approach: We developed a method that spatially parameterizes the confocal parameters in a previously established model for estimating the optical properties from the depth profiles of high-NA OCT.
Results: The proposed parametrization model was first evaluated on a set of intralipid phantoms and then validated using a low-NA objective in which cross-talk from the confocal parameters is negligible. We then utilize our spatially parameterized model to characterize optical property changes introduced by a tissue index matching process using a simple immersion agent, 2,2’-thiodiethonal.
Conclusions: Our approach improves the confidence of parameter estimation by reducing the degrees of freedom in the non-linear fitting model.
We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machine-learning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.
We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machine-learning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.
The three-dimensional reconstruction of large volumes of the human neural networks at cellular resolution is one of the biggest challenges of our days. Commonly, fine slices of samples marked with colorimetric techniques are individually imaged. This approach in addition to being time-consuming does not consider space cell organization, leading to loss of information. The aim of this work was to develop a methodology that allows analyzing the cytoarchitecture of the human brain in three dimensions at high resolution. In particular, we exploit the possibility of combining high-resolution 3D imaging techniques with clearing methodologies. We successfully integrate the SWITCH immunohistochemistry technique with the TDE clearing method to image a large volume of human brain tissue with two-photon fluorescence microscopy. In conclusion, this new approach enables to characterize large human brain specimens with high-resolution optical techniques, giving the possibility to expand the histological studies to the third dimension.
Quantitative and scalable whole-brain neuroanatomical mapping, with cellular resolution and molecular specificity, poses significant technological challenges. Indeed, a high image quality must be preserved reliably across the entire specimen and not only in a few representative volumes. On the other hand, robust and automated image analysis methods must be appropriately scalable to teravoxel datasets. Here, we present an experimental pipeline, involving tissue clearing, high-resolution light-sheet microscopy, volume registration to atlas, and deep learning strategies for image analysis, allowing the reconstruction of 3D maps of selected cell types in the whole mouse brain. We employed RAPID autofocusing [Silvestri et al., submitted] to keep the system sharply in focus throughout the entire mouse brain, without reducing the microscope throughput. Images were spatially anchored to reference atlas using semi-automatic tools (xNII family, http://www.nesys.uio.no). Finally, we used novel high-throughput tools for image processing, including deep learning strategies [Frasconi et al., 2014] to localize single neurons with high accuracy. By applying our pipeline to transgenically-labeled samples, we can produce an atlas of spatial distribution of genetically-defined cell types. Besides being a valuable reference for neurobiologists, these datasets can be used to build realistic simulations of neuronal functioning, such as in the Human Brain Project.
Remodeling processes associated with genetic and non-genetic cardiac diseases can cause alterations of electrical conduction and electro-mechanical dysfunction, eventually leading to arrhythmias. These alterations consist mainly in collagen deposition (fibrosis) and cellular disorganization (myofilament alignment), and their predictive models are often based on non-integrated and low-resolution information. Here, we combine advances in tissue clearing, immunostaining and high-resolution optical microscopy to reconstruct the three-dimensional organization of cardiac conduction system in the whole mouse heart. We developed a passive 2’2-thiodiethanol - Clarity protocol for clearing the heart and for achieving antibody penetration into the whole tissue. We simultaneously reconstructed the cellular organization stained by immunostaining and imaged the collagen distribution by second-harmonic generation deep in the cardiac tissue. A cytoarchitectonic analysis was applied to identify cells and to map myofilaments alignment in three dimensions, defining the conduction pathway of action potential propagation at intercellular level. We investigated the three-dimensional cytoarchitectonic remodeling in a transgenic mouse model of hypertrophic cardiomyopathy characterized by a severe degree of left ventricle hypertrophy and interstitial fibrosis. First, using a recently-developed ultra-fast optical system we mapped the propagation of electrical activity in whole diseased hearts. Then, we correlated the propagation maps with the pathological disorganization of myofilaments and collagen deposition using the three-dimensional high-resolution optical reconstruction. This innovative experimental approach will allow to dissect the morphological causes leading to alterations of electrical conduction and to electro-mechanical dysfunction, and, more generally, will represent a whole new paradigm for diagnostic and therapeutic investigations.
In this work, we employ an integrated label-free dual approach that combines Polarized
Light Imaging with Two-Photon Fluorescence Microscopy to study fiber orientations of
myelinated axons in fixed brain slices from different species.
Light-sheet microscopy enables whole mouse brain imaging in association with clearing methodologies. Here, we present a pipeline for optimal investigation of the vascular component, which offers improved image quality for morphological analysis.
Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.
Ludovico Silvestri, Nikita Rudinskiy, Marco Paciscopi, Marie Caroline Müllenbroich, Irene Costantini, Leonardo Sacconi, Paolo Frasconi, Bradley Hyman, Francesco Pavone
Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information.
Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution.
The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.
Chemical clearing of fixed tissues is becoming a key instrument for the three-dimensional reconstruction of macroscopic tissue portions, including entire organs. Indeed, the growing interest in this field has both triggered and been stimulated by recent advances in high-throughput microscopy and data analysis methods, which allowed imaging and management of large samples. The strong entanglement between clearing methods and imaging technology is often overlooked, as typical classification of the former is based only on the chemicals used. Here, we review the recent literature in the field, proposing a taxonomy of clearing techniques based on their mating with the major high-throughput microscopies. We hope that this application-oriented classification can help researchers to find the protocol best suited to their experiment among the many present in the literature.
Large volumes imaging with microscopic resolution is limited by light scattering. In the last few years based on refractive index matching, different clearing approaches have been developed. Organic solvents and water-based optical clearing agents have been used for optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a highly expensive refractive index matching solution limiting practical applicability. In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for CLARITY. The potential of this method has been explored by imaging a whole transgenic mouse brain with the light sheet microscope. Moreover we apply this technique also on blocks of dysplastic human brain tissue transformed with CLARITY and labeled with different antibody. This clearing approach significantly expands the application of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in mouse and human brain.
M. Caroline Müllenbroich, Ludovico Silvestri, Leonardo Onofri, Irene Costantini, Marcel van’t Hoff, Leonardo Sacconi, Giulio Iannello, Francesco S. Pavone
Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.
Nowadays, there are several imaging techniques offering a complementary approach to visualize intact neural networks on large areas. Each of those offers a different strategy and furnish complementary information on the role of neural components. We will describe different approaches enabling to move from single neuron details to whole brain imaging, connecting short range structural information to long range one. In particular, some examples of correlative microscopies, combining linear and non linear techniques will also be described.
Light scattering inside biological tissue is a limitation for large volumes imaging with microscopic resolution. Based on
refractive index matching, different approaches have been developed to reduce scattering in fixed tissue. High refractive
index organic solvents and water-based optical clearing agents, such as Sca/e, SeeDB and CUBIC have been used for
optical clearing of entire mouse brain. Although these methods guarantee high transparency and preservation of the
fluorescence, though present other non-negligible limitations. Tissue transformation by CLARITY allows high
transparency, whole brain immunolabelling and structural and molecular preservation. This method however requires a
highly expensive refractive index matching solution limiting practical applicability to large volumes.
In this work we investigate the effectiveness of a water-soluble clearing agent, the 2,2'-thiodiethanol (TDE) to clear
mouse and human brain. TDE does not quench the fluorescence signal, is compatible with immunostaining and does not
introduce any deformation at sub-cellular level. The not viscous nature of the TDE make it a suitable agent to perform
brain slicing during serial two-photon (STP) tomography. In fact, by improving penetration depth it reduces tissue
slicing, decreasing the acquisition time and cutting artefacts. TDE can also be used as a refractive index medium for
CLARITY. The potential of this method has been explored by imaging blocks of dysplastic human brain transformed
with CLARITY, immunostained and cleared with the TDE. This clearing approach significantly expands the application
of single and two-photon imaging, providing a new useful method for quantitative morphological analysis of structure in
mouse and human brain.
Brain imaging is becoming an important field in the frame of the neurophotonics in correlations with other medical ones in neuroscience studying functional and morphological aspects. In this presentation an overview on multi photon imaging of the brain will be presented, together with innovative aspects related to big area imaging and correlative microscopy approaches. Multiphoton imaging applications will be described together with methods to improve the penetration depth and obtain large area detection, or correlating functional aspects in vivo on single neuron with large area, even on whole brain, morphological aspects. Connecting super resolution features at the nanometer level with micro, meso and macroscopic architectures is in fact one of the challenging aspects to understand brain functioning.
One of the unique features of the brain is that its activity cannot be framed in a single spatio-temporal scale, but rather spans many orders of magnitude both in space and time. A single imaging technique can reveal only a small part of this complex machinery. To obtain a more comprehensive view of brain functionality, complementary approaches should be combined into a correlative framework. Here, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, taking advantage of blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living thy1-GFP-M mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from the apical portion, the whole pyramidal neuron can then be segmented. The correlative approach presented here allows contextualizing within a three-dimensional anatomic framework the neurons whose dynamics have been observed with high detail in vivo.
KEYWORDS: Microscopy, In vivo imaging, Neurons, Brain, Confocal microscopy, Neuroimaging, Electron microscopy, Axons, Two photon excitation microscopy, Luminescence
Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy,
combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can
be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron
imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting
from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.
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