Two Photon structural and functional imaging

We exploit the high-resolution and penetration depth achievable with Two-Photon Fluorescence Microscopy (TPFM) to perform both structural and functional analysis on different animal models, and mesoscopic reconstruction of human brain tissue. 


We perform in vivo two-photon imaging on transgenic mice expressing the Green Fluorescent Protein (GFP) in excitatory neurons. This technique allows to longitudinally study both presynaptic (axonal fibers and axonal varicosities) and postsynaptic (dendrites and dendritic spines) neuronal structures in the healthy and injured brain. In detail, we investigate the structural remodeling of pyramidal apical dendrites and axons at increasing distances from stroke core in the mouse cortex during spontaneous recovery and different rehabilitative approaches. Moreover, we perform two-photon imaging on awake mice expressing a calcium indicator (GCaMP6f) in excitatory neurons while the animal is performing a motor task. This allows us to investigate alteration in motor-evoked cortical functionality with single-cell resolution and to characterize the effect of cortical stroke and rehabilitation paradigms on functional plasticity. 


TPFM imaging is performed in zebrafish larvae expressing GCaMP6s at pan-neuronal level. The capability of imaging the whole brain volume at different developmental stages allows mapping development of circuits and following the temporal evolution of functional systems, such as those involved in sensorial perception and motor response generation. Structural mapping also allows evaluation of the degree of variation among different individuals. Functional imaging is employed to explore the onset of patterns of activity linked with oscillatory and spontaneous circuits, for example related with circadian rhythms and other periodic phenomena. 


Mesoscopic reconstruction obtained with TPFM of different portions of bulk samples of the human cortex from control subjects and patients with malformations of cortical development (MCDs) are produced to study the anatomical organization of neurons in three-dimensions with high resolution. 

A SWITCH-TDE clearing approach is used to obtain good transparency and multi-antigen staining of different kinds of tissues (child, adult, and elderly subject). A machine-learning based strategy able to perform an automated neuronal segmentation is used to study the distribution of cells volume and density through the cortical layers. In parallel, to obtain inclusive connectomics maps of the human brain, high-resolution mesoscopic reconstruction of myelinated fibers in 3D are performed on brain slices previously imaged with fMRI and 3D-PLI. To discriminate against different adjacent myelinated axons we developed MAGIC (Myelin Autofluorescence imaging by Glycerol Induced Contrast enhancement), a novel, simple method to perform label-free fluorescence imaging of myelinated fibers based on glycerol embedding of the tissue. This multi-modal analysis enables to study the fiber organization of the human brain in three-dimension.

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