Eventi di rinnovo e risposta lineare nella turbolenza debole dei cristalli liquidi nematici

In questa tesi si sono studiate sperimentalmente le fluttuazioni e la risposta a stimoli esterni di un campione di cristallo liquido nematico (MBBA) in un regime di turbolenza debole. Per raggiungere questo stato si è messo il cristallo liquido tra due piastre di vetro ricoperte di ossido di indio ed è stata applicata una differenza di potenziale ai capi di queste. Quando il voltaggio supera un valore critico si osservano delle instabilità elettroconvettive analoghe a quelle di Rayleigh-Bénard. Il pattern formato dai rulli di convezione è facilmente osservabile mediante un microscopio polarizzatore a causa della birifrangenza del cristallo liquido nematico, per cui l’intensità della luce trasmessa dipende punto per punto dal campo di orientazione locale, che a sua volta è legato al campo idrodinamico delle velocità. All’aumentare della tensione applicata nascono nel pattern dei difetti (dislocazioni) che mostrano diversi comportamenti dinamici: possono derivare, oscillare, nascere e morire. Per queste caratteristiche l’elettroidroconvezione dei cristalli liquidi nematici è un paradigma per molti sistemi complessi, caratterizzati dall’emergenza di strutture auto-organizzate e dalla loro interazione. Questi sistemi vivono fuori dall’equilibrio termodinamico e possono mostrare una rottura delle tradizionali relazioni di fluttuazione e dissipazione ed in generale della teoria della risposta lineare. Di questa teoria è stata tuttavia recentemente proposta una generalizzazione, basata su un modello fenomenologico per sistemi complessi. Questo modello ipotizza che la dinamica del sistema sia data dall’alternarsi di momenti di forte attività dinamica completamente scorrelati fra loro, detti eventi di rinnovo, e di zone di relativa calma dette regioni laminari. La complessità si manifesta nella distribuzione dei tempi di attesa tra un evento e l’altro, che è caratterizzata da un andamento a potenza inversa 1/t^mu e non esponenziale: il parametro mu può quindi caratterizzare quantitativamente il sistema. La teoria prevede poi che le fluttuazioni generate da un processo di questo tipo hanno uno spettro con un andamento 1/f^eta per f->0, con eta = 3 -mu. Il lavoro sperimentale svolto in questa tesi si è articolato in tre fasi, durante le quali si è osservata una variabile globale, cioè l’intensità luminosa trasmessa dal cristallo liquido, sotto diverse condizioni. Anzitutto si sono studiate le fluttuazioni del sistema al variare del voltaggio ed osservando, per un ampio range della tensione applicata, uno spettro del tipo 1/f^eta con 1<eta<2. Per valutare con più precisione questo parametro sono state effettuate analisi con tecniche più sofisticate, come la DFA, l’entropia di diffusione e la SDA. Successivamente si è studiata il rilassamento della luminosità totale ad un valore stazionario in seguito ad un brusco cambiamento del voltaggio applicato. I rilassamenti osservati sono incompatibili con la teoria della risposta lineare tradizionale ma in ottimo accordo con la sua generalizzazione per sistemi dominati da eventi di rinnovo. Come ultima cosa è stata studiata la risposta del sistema a modulazioni dell’ampiezza del voltaggio applicato usando due diversi tipi di perturbazione: una sinusoide a bassa frequenza e un segnale “complesso”, costituito dalle fluttuazioni 1/f^eta del sistema stesso precedentemente registrate. Si è valutata la correlazione tra le fluttuazioni del sistema e la modulazione per entrambi i tipi di perturbazione e nel caso di perturbazione complessa si è studiato come questa è in grado di influenzare le proprietà frattali del sistema

Towards a comprehensive understanding of brain machinery by correlative microscopy.

Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation

Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain.

Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. (C) 2012 Optical Society of Americ

Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. 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 quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

Large-scale automated identification of mouse brain cells in confocal light sheet microscopy images

Motivation: Recently, confocal light sheet microscopy has enabled high-throughput acquisition of whole mouse brain 3D images at the micron scale resolution. This poses the unprecedented challenge of creating accurate digital maps of the whole set of cells in a brain. Results: We introduce a fast and scalable algorithm for fully automated cell identification. We obtained the whole digital map of Purkinje cells in mouse cerebellum consisting of a set of 3D cell center coordinates. The method is accurate and we estimated an F(1) measure of 0.96 using 56 representative volumes, totaling 1.09 GVoxel and containing 4138 manually annotated soma centers. Availability and implementation: Source code and its documentation are available at http://bcfind.dinfo.unifi.it/. The whole pipeline of methods is implemented in Python and makes use of Pylearn2 and modified parts of Scikit-learn. Brain images are available on request. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

ADVANCED OPTICAL TECHNIQUES TO EXPLORE BRAIN STRUCTURE AND FUNCTION

Understanding brain structure and function, and the complex relationships between them, is one of the grand challenges of contemporary sciences. Thanks to their flexibility, optical techniques could be the key to explore this complex network. In this manuscript, we briefly review recent advancements in optical methods applied to three main issues: anatomy, plasticity and functionality. We describe novel implementations of light-sheet microscopy to resolve neuronal anatomy in whole fixed brains with cellular resolution. Moving to living samples, we show how real-time dynamics of brain rewiring can be visualized through two-photon microscopy with the spatial resolution of single synaptic contacts. The plasticity of the injured brain can also be dissected through cutting-edge optical methods that specifically ablate single neuronal processes. Finally, we report how nonlinear microscopy in combination with novel voltage sensitive dyes allow optical registrations of action potential across a population of neurons opening promising prospective in understanding brain functionality. The knowledge acquired from these complementary optical methods may provide a deeper comprehension of the brain and of its unique features

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