Biology subjects

Costantini, I.

Publications and source records attributed to Costantini, I..

17 recordsLinked to original sources

bioRxiv2025

Anisotropic light propagation in human brain white matter

SignificanceAccurate modeling of light diffusion in the human brain is crucial for applications in optogenetics and spectroscopy diagnostic techniques. White matter tissue is composed of myelinated axon bundles, suggesting the occurrence of enhanced light diffusion along their local orientation direction, which however has never been characterized experimentally. Existing diffuse optics models assume isotropic properties, limiting their accuracy. AimWe aim to characterize the anisotropic scattering properties of human white matter tissue by directly measuring its tensor scattering components along different directions, and to correlate them with the local axon fiber orientation. ApproachUsing a time- and space-resolved setup, we image the transverse propagation of diffusely reflected light across two perpendicular directions in a ex vivo human brain sample. Local fiber orientation is independently determined using light sheet fluorescence microscopy (LSFM). ResultsThe directional dependence of light propagation in organized myelinated axon bundles is characterized via Monte Carlo (MC) simulations accounting for a tensor scattering coefficient, revealing a lower scattering rate parallel to the fiber orientation. The effects of white matter anisotropy are further assessed by simulating a typical time-domain near-infrared spectroscopy measurement in a four-layer human head model. ConclusionsThis study provides a first characterization of the anisotropic scattering properties in ex vivo human white matter, highlighting its direct correlation with axon fiber orientation, and opening to the realization of quantitatively accurate anisotropy-aware human head 3D meshes for diffuse optics applications.

neuroscience↗

bioRxiv2025

Myelinated fiber labeling and orientation mapping of the human brain with light-sheet fluorescence microscopy

The convoluted network of myelinated fibers that supports behavior, cognition, and sensory processing in the human brain is the source of its extraordinary complexity. Advancements in tissue optical clearing, 3D fluorescence microscopy, and automated image analysis have enabled unprecedented insights into the architecture of these networks. Here, we investigate the multiscale organization of myelinated fibers in human brain tissue from the brainstem, Brocas area, hippocampus, and primary visual cortex by exploiting a specific fiber staining method, light-sheet fluorescence microscopy (LSFM), and an advanced spatial orientation analysis tool. Using an optimized protocol that integrates tissue clearing with the lipophilic DiD probe to achieve uniform and deep myelinated fiber labeling, we generate micrometerresolution volumetric reconstructions of multiple brain regions through an inverted LSFM. Automated image processing, employing unsupervised 3D multiscale Frangi filters, provides orientation distribution functions and local orientation dispersion maps. This enables precise characterization of the directionality of white matter bundles, linking mesoscopic structural properties to orientation details computed at the native micrometric resolution of the LSFM apparatus. The presented workflow illustrates a robust platform for large-scale, high-resolution brain mapping, which may facilitate the investigation of pathological alterations with unparalleled spatial resolution and, furthermore, the validation of other neuroimaging modalities.

neuroscience↗

bioRxiv2024

Quantitative cytoarchitectural phenotyping of deparaffinized human brain tissues

Advanced 3D imaging techniques and image segmentation and classification methods can profoundly transform biomedical research by offering deep insights into the cytoarchitecture of the human brain in relation to pathological conditions. Here, we propose a comprehensive pipeline for performing 3D imaging and automated quantitative cellular phenotyping on Formalin-Fixed Paraffin-Embedded (FFPE) human brain specimens, a valuable yet underutilized resource. We exploited the versatility of our method by applying it to different human specimens from both adult and pediatric, normal and abnormal brain regions. Quantitative data on neuronal volume, ellipticity, local density, and spatial clustering level were obtained from a machine learning-based analysis of the 3D cytoarchitectural organization of cells identified by different molecular markers in two subjects with malformations of cortical development (MCD). This approach will grant access to a wide range of physiological and pathological paraffin-embedded clinical specimens, allowing for volumetric imaging and quantitative analysis of human brain samples at cellular resolution. Possible genotype-phenotype correlations can be unveiled, providing new insights into the pathogenesis of various brain diseases and enlarging treatment opportunities.

neuroscience↗

bioRxiv2023

Segmentation of supragranular and infragranular layers in ultra-high resolution 7T ex vivo MRI of the human cerebral cortex

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Lever-aging 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 {micro}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.

neuroscience↗

bioRxiv2023

Microplastics reach the brain and interfere with honey bee cognition

Scientific research exploring the impact of microplastics (MPs) in terrestrial systems is still at an early stage but has already confirmed that exposure to plastics leads to various detrimental health effects in several organisms. Although recent studies have shown the toxicological effects of single MP polymers on honey bees, the effects of different polymer combinations and their consequences on cognitive and behavioural performance remain unknown. To fill this knowledge gap, we investigated the effects of MPs, both individually and in combination, on the cognitive abilities of the honey bee Apis mellifera. We evaluated the acute oral toxicity of Polystyrene (PS) and Plexiglass (PMMA) MPs, as well as a combination of the two (MIX), at three different concentrations (0.5, 5 and 50 mg/L-1) and analysed their effects on sucrose responsiveness and appetitive olfactory learning and memory. We also explored whether these MPs could reach and accumulate in the insect brain using Two-Photon Fluorescence Microscopy (TPFM) in combination with an optimized version of the DISCO clearing technique. The results revealed that PS reduced the responsiveness of foragers to sucrose, whereas PMMA had no significant impact; however, the combination of PMMA and PS had a pronounced negative effect on sucrose responsiveness. In addition, both PMMA and PS, as well as MIX, impaired bee learning formation and memory retrieval, with PS exhibiting the most severe effects. Regarding our brain imaging analysis performed with TFPM, we found that after only three days of oral exposure, MPs could penetrate and accumulate in the brain. These results raise concerns about the potential mechanical, cellular, and biochemical damage that MPs may cause to the central nervous system.

animal behavior and cognition↗

bioRxiv2022

Fiber enhancement and 3D orientation analysis inlabel-free two-photon fluorescence microscopy

3D fluorescence microscopy can be profitably used in combination with tissue clearing and myelin labeling techniques for investigating the human brain myeloarchitectonics with sub-micrometric resolution, and for providing detailed histological reference data for the validation of diffusion magnetic resonance imaging (dMRI) tractography. Differently from state-of-the-art polarimetry-based neuroimaging modalities such as 3D polarized light imaging and 3D polarized-sensitive optical coherence tomography, the quantitative histological analysis of cortical and white matter fiber architectures from fluorescence microscopy images requires the estimation of fiber orientations via image processing techniques, as Fourier or structure tensor analysis. However, these can be error-prone as they are not able to detect myelinated fibers in surrounding brain tissue. Here we present a novel image processing pipeline built around a 3D Frangi filter able to enhance fiber structures of varying diameters in tiled brain section reconstructions of arbitrary size, and to produce accurate 3D fiber orientation maps in both grey and white matter regions, while preserving the native resolution of the employed microscopy system. The developed software tool also features the estimation of fiber orientation distribution functions which, in view of their widespread use in the neuroimaging community, may favor a multimodal comparison with brain connectivity data obtained from the aforementioned label-free optical modalities, and the validation of modern dMRI-based tractography. The proposed image processing pipeline was applied to brain samples treated according to a novel label-free preparation technique capable of enhancing the autofluorescence of myelinated fiber axons, thus demonstrating the feasibility of single fiber orientation analyses in fluorescence microscopy without the need for exogenous staining.

bioengineering↗

bioRxiv2021

A multimodal imaging and analysis pipeline for creating a cellular census of the human cerebral cortex

Cells are not uniformly distributed in the human cerebral cortex. Rather, they are arranged in a regional and laminar fashion that span a range of scales. Here we demonstrate an innovative imaging and analysis pipeline to construct a reliable cell census across the human cerebral cortex. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system.

neuroscience↗

bioRxiv2021

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

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.

neuroscience↗

bioRxiv2021

Exploring the human cerebral cortex using confocal microscopy

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.

neuroscience↗

bioRxiv2021

A Paradigm Free Regularization Approach to Recover Brain Activation from Functional MRI Data

ContextFunctional MRI is a non-invasive imaging technique that provides an indirect view into brain activity, via the blood-oxygen-level-dependent (BOLD) response. In particular, resting-state fMRI poses challenges to the recovery of brain activity without prior knowledge on the experimental paradigm, as it is the case for task-fMRI. Conventional methods to infer brain activity from the fMRI signals, for example the general linear model (GLM), require the knowledge of the experimental paradigm to define regressors and estimate the contribution of each voxels time course to the task. To overcome this limitation, approaches to deconvolve the BOLD response and recover the underlying neural activation without a priori information on the task have been proposed. State-of-the-art techniques, and in particular the Total Activation (TA), formulates the deconvolution as an optimization problem with decoupled spatial and temporal regularization terms. This increases the number of hyperparameters to be set and requires an optimization strategy that alternates between the constraints. ApproachIn this work, we propose a paradigm-free regularization algorithm named Paradigm-Free fMRI (PF-fMRI) that is applied on the 4-D fMRI image, acting simultaneously in the 3-D space and 1-D time dimensions. Based on the idea that large image variations should be preserved as they occur during brain activation, whereas small variations considered as noise should be removed, the PF-fMRI applies an anisotropic regularization, thus recovering the location and the duration of brain activation. ResultsUsing the experimental paradigm as ground truth, the PF-fMRI is validated on synthetic and real task-fMRI data from 51 subjets, and its performance is compared to the TA. Results show higher correlations of the recovered time-courses with the ground truth compared to the TA and lower computational times. In addition, we show that the PF-fMRI recovers activity that agrees with the GLM, without requiring or using any knowledge of the experimental paradigm.

neuroscience↗

bioRxiv2020

Fast volumetric mapping of human brain slices

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 machinelearning 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.

biophysics↗

bioRxiv2020

3D imaging and morphometry of the heart capillary system in spontaneously hypertensive rats and normotensive controls

Systemic arterial hypertension is a highly prevalent chronic disease associated with hypertensive cardiomyopathy. One important feature of this condition is remodelling of intramural small coronary arteries and arterioles. Here, we investigated the implications of this remodelling in the downstream vascular organization, in particular at the capillary level. We used Spontaneously Hypertensive Rats (SHR) exhibiting many features of the human hypertensive cardiomyopathy. We generated 3D high-resolution mesoscopic reconstructions of the entire network of SHR hearts combining gel-based fluorescent labelling of coronaries with a CLARITY-based tissue clearing protocol. We performed morphometric quantification of the capillary network over time to assess capillary diameter, linear density, and angular dispersion. In SHRs, we found significant remodelling of the capillary network density and dispersion. SHR capillary density is increased in both ventricles and at all ages, including before the onset of systemic hypertension. This result suggests that remodelling occurs independently from the onset of systemic hypertension and left ventricular hypertrophy. On the contrary, capillary angular dispersion increases with time in SHR. Consistently, our multi-modal imaging underlined a strong correlation between vascular dispersion and cellular disarray. Together our data show that 3D high-resolution reconstruction of the capillary network can unveil anatomic signatures in both physiological and pathological cardiac conditions, thus offering a reliable method for integrated quantitative analyses.

physiology↗

bioRxiv2020

A combined pipeline for quantitative analysis of human brain cytoarchitecture

The 3D analysis of the human brain architecture at cellular resolution is still a big challenge. In this work, we propose a pipeline that solves the problem of performing neuronal mapping in large human brain samples at micrometer resolution. First, we introduce the SWITCH/TDE protocol: a robust methodology to clear and label human brain tissue. Then, we implement the 2.5D method based on a Convolutional Neural Network, to automatically detect and segment all neurons. Our method proved to be highly versatile and was applied successfully on specimens from different areas of the cortex originating from different subjects (young, adult and elderly, both healthy and pathological). We quantitatively evaluate the density and, more importantly, the mean volume of the thousands of neurons identified within the specimens. In conclusion, our pipeline makes it possible to study the structural organization of the brain and expands the histopathological studies to the third dimension.

neuroscience↗

bioRxiv2020

MAGIC: A label-free fluorescence method for 3D high-resolution reconstruction of myelinated fibers in large volumes

Analyzing the structure of neuronal fibers with single axon resolution, in large volumes, remains an unresolved challenge in connectomics. Here, we propose MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a simple tissue preparation method to perform label-free fluorescence imaging of myelinated fibers. We demonstrate its broad applicability by performing mesoscopic reconstruction at sub-micron resolution of mouse, rat, monkey, and human brain samples and by quantifying the different fiber organization in Control and Reeler mouses hippocampal sections.

neuroscience↗

bioRxiv2017

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.

neuroscience↗

bioRxiv2017

Whole-brain vasculature reconstruction at the single capillary level

The distinct organization of the brains 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.

neuroscience↗

bioRxiv2017

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.

bioengineering↗