Antonino Paolo Di Giovanna

A versatile clearing agent for multi-modal brain imaging

Extensive mapping of neuronal connections in the central nervous system requires high-throughput µm-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multi-modal optical techniques. Here, we introduce a versatile brain clearing agent (2,2′-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.

Whole-Brain Vasculature Reconstruction at the Single Capillary Level

The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.

High-fidelity functional and structural whole-brain imaging with Bessel-beam light-sheet microscopy (Conference Presentation)

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience and is particularly well suited to image the entire brain with high frame rates at single cell resolution. On the one hand, LSM is employed in combination with tissue clearing methods like CLARITY which allows for the reconstruction of neuronal or vascular anatomy over cm-sized samples. On the other hand, LSM has been paired with intrinsically transparent samples for real-time recording of neuronal activity with single cell resolution across the entire brain, using calcium indicators like GCaMP6. Despite its intrinsic advantages in terms of high imaging speed and reduced photobleaching, LSM is very sensitive to residual opaque objects present in the sample, which cause dark horizontal stripes in the collected images. In the best case, these artefacts obscure the features of interest in structural imaging; in the worst case, dynamic shadowing introduced by red blood cells significantly alters the fluorescence signal variations related to neuronal activity. We show how the use of Bessel beams in LSM can dramatically reduce such artefacts even in conventional one-sided illumination schemes, thanks to their “self-healing” properties. On the functional side, Bessel-beam LSM allows recording neuronal activity traces without any disturbing flickering caused by the movement of red blood cells. On the structural side, our proposed method is capable of obtaining anatomical information across the entire volume of whole mouse brains allowing tracing blood vessels and neuronal projections also in poorly cleared specimens.

Optimal staining and clearing protocol for whole mouse brain vasculature imaging with light-sheet microscopy

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.

Mapping the quantitative cytoarchitecture of the whole mouse brain by light-sheet microscopy and digital brain atlasing (Conference Presentation)

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.

Rehabilitation Restores Cortical Activation Profiles And Stabilizes Synaptic Contacts After Stroke

Neuro-rehabilitative therapy is the most effective treatment for recovering motor deficits in stroke patients. Nevertheless, the neural basis of recovery associated with rehabilitative intervention is debated. Here, we addressed the multiple facets of cortical remodeling induced by rehabilitative therapy. By longitudinal imaging of cortical activity while training, we demonstrated progressive attenuation of motor map dedifferentiation and rise of a more intense and fast-rising calcium response in the peri-infarct area during movement execution. Coupling of the spared cortex to the injured hemisphere was reinforced by rehabilitation, as demonstrated by our all-optical approach. Alongside, profound angiogenic response accompanied the stabilization of peri-infarct micro-circuitry at the synaptic level. The present work, by combining optical tools of visualization and manipulation of neuronal activity, provides the first in vivo evidence of the mechanism of rehabilitation in shaping cortical plasticity.Rehabilitation is the most effective treatment for promoting the recovery of motor deficits after stroke. Despite its importance, the processes associated with rehabilitative intervention are poorly understood. One of the most challenging experimental goals is to unambiguously link specific circuit changes induced by rehabilitation in recovering animals to improved behavior. Here, we investigate which facets of cortical remodeling are induced by rehabilitative therapy after stroke by combining optical imaging and manipulation tools. We demonstrate the progressive restoration of cortical motor maps and of cortical activity in parallel with the reinforcement of inter-hemispheric connectivity after rehabilitation. Furthermore, we reveal that the increase in vascular density goes along with the stabilization of peri-infarct neural circuitry at synaptic level. The present work provides the first evidences that rehabilitation is sufficient to promote the combined recovery of distinct structural and functional features distinctive of healthy neuronal networks. We believe that understanding the plastic changes induced by rehabilitation will lead to more effective interventions to improve recovery after stroke.

Increasing sensitivity and accuracy of brain-wide quantitative studies in light-sheet microscopy

Light-sheet microscopy (LSM) has proven a useful tool in neuroscience to image whole brains with high frame rates at cellular resolution. LSM is employed either in combination with tissue clearing to reconstruct the cyto-architecture over the entire mouse brain or with intrinsically transparent samples like zebrafish larvae for functional imaging. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity structural data. Furthermore, using Bessel beams, we demonstrate a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Our results demonstrate the contamination of data by systematic and random errors through Gaussian illumination and furthermore quantify the increase in fidelity of such data when using Bessel beams.

RAPID: Real-time image-based autofocus for all wide-field optical microscopy systems

Autofocus methods used in biomicroscopy are either based on the search of an optimal focus position – which requires suspending data collection during the optimization process – or on the continuous monitoring of the position of a fiducial plane – which may not coincide with the sample itself. Here, we introduce RAPID (Rapid Autofocus via Pupil-split Image phase Detection), a method for real-time image-based focus stabilization, applicable in all wide-field microscopy systems. We demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to light-sheet microscopy. RAPID provides a universal autofocus solution for automated microscopy, and enables quantitative assays otherwise impossible in a standard microscope, such as 3D tracking of fast-moving organisms.

Rehabilitation-triggered cortical plasticity after stroke: in vivo imaging at multiple scales (Conference Presentation)

Neurorehabilitation protocols based on the use of robotic devices provide a highly repeatable therapy and have recently shown promising clinical results. Little is known about how rehabilitation molds the brain to promote motor recovery of the affected limb. We used a custom-made robotic platform that provides quantitative assessment of forelimb function in a retraction test. Complementary imaging techniques allowed us to access to the multiple facets of robotic rehabilitation-induced cortical plasticity after unilateral photothrombotic stroke in mice Primary Motor Cortex (Caudal Forelimb Area - CFA). First, we analyzed structural features of vasculature and dendritic reshaping in the peri-infarct area with two-photon fluorescence microscopy. Longitudinal analysis of dendritic branches and spines of pyramidal neurons suggests that robotic rehabilitation promotes the stabilization of peri-infarct cortical excitatory circuits, which is not accompanied by consistent vascular reorganization towards pre-stroke conditions. To investigate if this structural stabilization was linked to functional remapping, we performed mesoscale wide-field imaging on GCaMP6 mice while performing the motor task on the robotic platform. We revealed temporal and spatial features of the motor-triggered cortical activation, shining new light on rehabilitation-induced functional remapping of the ipsilesional cortex. Finally, by using an all-optical approach that combines optogenetic activation of the contralesional hemisphere and wide-field functional imaging of peri-infarct area, we dissected the effect of robotic rehabilitation on inter-hemispheric cortico-cortical connectivity.