Dynamic processes are ubiquitous in biological systems: the transport of organelles, proteins and cargoes in the micron-sized heterogeneous cellular environment mainly occurs by Brownian diffusion, while directional flow or drift phenomena contribute to enhance the diffusion-mediated intracellular trafficking and are responsible for the delivery of blood, nutrients and signaling molecules on the larger scale of whole tissues and organs. Motivated by the relevance of transport phenomena in several fields ranging from cell biology to immunology, I adopt and extend the approach of Fluorescence Image Correlation Spectroscopy to investigate diffusive and directional transport processes from the single-cell level up to whole microcirculatory systems. Dynamic transport parameters are quantified by the spatial and/or temporal correlation, in the direct or the reciprocal Fourier space, of the raster-scanned images acquired in-vivo by fluorescence (or reflectance) confocal microscopy. In this work, I focus at first on the measurement of flow velocities in geometrically complex microcirculatory networks, with the development of a novel image-processing method that I have called FLICS or FLow Image Correlation Spectroscopy. FLICS has the peculiarity of exploiting a single raster-scanned xy-image, acquired by detecting the signal of bright, sparse flowing objects (e.g., erythrocytes): by the Cross Correlation Function (CCF) of the fluorescence fluctuations detected in pairs of columns of the image, both the modulus and the direction of the flow velocity can be recovered and mapped, with single-capillary sensitivity and sub-second time resolution, in the whole vessel pattern within the imaged field of view. I derive the explicit analytical expression of the CCF for both two- and three- dimensional flow velocity vectors and, by the approximation of negligible Brownian diffusion, I refine the data-analysis protocol to optimize the flow speed measurement over extended circulatory networks. I validate the FLICS theoretical framework in systems of increasing complexity and I finally apply the method to the characterization of the sinusoidal blood flow in the intricate murine hepatic microcirculation. On the smaller single-cell spatial scale, I successively employ live-cell time-lapse confocal reflectance microscopy and image correlation to investigate the intracellular transport of branched, star-like nanoparticles (GNSs, or Gold NanoStars). Different transport mechanisms, spanning from Brownian diffusion to (sub-)ballistic super-diffusion, are revealed by Temporal and Spatio-Temporal Image Correlation Spectroscopy on the tens-of-seconds timescale. By combining these findings with numerical simulations and with a Bayesian (Hidden Markov Model based) analysis of single particle tracking data, I ascribe the super-diffusive, sub-ballistic behavior of the GNSs dynamics to a two-state switch between diffusion in the cytoplasm and molecular motor-mediated active transport along cytoskeletal filaments. I derive therefore a novel analytical theoretical framework for the investigation of intermittent transport by Fourier-space Image Correlation Spectroscopy (kICS). Besides evaluating on simulated kICS correlation functions the influence of all the dynamic parameters and of the transition rates between the diffusive and the active transport regimes, I derive whole-cell maps for the parameters underlying the GNSs intracellular dynamics. Notably, the method is capable of identifying the simplest transport mode that accurately describes the experimental data, without any prior assumption on its Brownian or super-diffusive nature. The results obtained here for the subcellular trafficking of gold nanostars will be of help in the rational design of the drug delivery and photo-thermal therapy applications of anisotropic gold nanoparticles.

(2015). Correlazione di Immagini per lo Studio di Processi Dinamici in Sistemi Biologici. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Correlazione di Immagini per lo Studio di Processi Dinamici in Sistemi Biologici

BOUZIN, MARGAUX
2015

Abstract

Dynamic processes are ubiquitous in biological systems: the transport of organelles, proteins and cargoes in the micron-sized heterogeneous cellular environment mainly occurs by Brownian diffusion, while directional flow or drift phenomena contribute to enhance the diffusion-mediated intracellular trafficking and are responsible for the delivery of blood, nutrients and signaling molecules on the larger scale of whole tissues and organs. Motivated by the relevance of transport phenomena in several fields ranging from cell biology to immunology, I adopt and extend the approach of Fluorescence Image Correlation Spectroscopy to investigate diffusive and directional transport processes from the single-cell level up to whole microcirculatory systems. Dynamic transport parameters are quantified by the spatial and/or temporal correlation, in the direct or the reciprocal Fourier space, of the raster-scanned images acquired in-vivo by fluorescence (or reflectance) confocal microscopy. In this work, I focus at first on the measurement of flow velocities in geometrically complex microcirculatory networks, with the development of a novel image-processing method that I have called FLICS or FLow Image Correlation Spectroscopy. FLICS has the peculiarity of exploiting a single raster-scanned xy-image, acquired by detecting the signal of bright, sparse flowing objects (e.g., erythrocytes): by the Cross Correlation Function (CCF) of the fluorescence fluctuations detected in pairs of columns of the image, both the modulus and the direction of the flow velocity can be recovered and mapped, with single-capillary sensitivity and sub-second time resolution, in the whole vessel pattern within the imaged field of view. I derive the explicit analytical expression of the CCF for both two- and three- dimensional flow velocity vectors and, by the approximation of negligible Brownian diffusion, I refine the data-analysis protocol to optimize the flow speed measurement over extended circulatory networks. I validate the FLICS theoretical framework in systems of increasing complexity and I finally apply the method to the characterization of the sinusoidal blood flow in the intricate murine hepatic microcirculation. On the smaller single-cell spatial scale, I successively employ live-cell time-lapse confocal reflectance microscopy and image correlation to investigate the intracellular transport of branched, star-like nanoparticles (GNSs, or Gold NanoStars). Different transport mechanisms, spanning from Brownian diffusion to (sub-)ballistic super-diffusion, are revealed by Temporal and Spatio-Temporal Image Correlation Spectroscopy on the tens-of-seconds timescale. By combining these findings with numerical simulations and with a Bayesian (Hidden Markov Model based) analysis of single particle tracking data, I ascribe the super-diffusive, sub-ballistic behavior of the GNSs dynamics to a two-state switch between diffusion in the cytoplasm and molecular motor-mediated active transport along cytoskeletal filaments. I derive therefore a novel analytical theoretical framework for the investigation of intermittent transport by Fourier-space Image Correlation Spectroscopy (kICS). Besides evaluating on simulated kICS correlation functions the influence of all the dynamic parameters and of the transition rates between the diffusive and the active transport regimes, I derive whole-cell maps for the parameters underlying the GNSs intracellular dynamics. Notably, the method is capable of identifying the simplest transport mode that accurately describes the experimental data, without any prior assumption on its Brownian or super-diffusive nature. The results obtained here for the subcellular trafficking of gold nanostars will be of help in the rational design of the drug delivery and photo-thermal therapy applications of anisotropic gold nanoparticles.
COLLINI, MADDALENA
Fluorescence Microscopy, Image Correlation Spectroscopy
FIS/07 - FISICA APPLICATA (A BENI CULTURALI, AMBIENTALI, BIOLOGIA E MEDICINA)
English
1-dic-2015
FISICA E ASTRONOMIA - 30R
28
2014/2015
open
(2015). Correlazione di Immagini per lo Studio di Processi Dinamici in Sistemi Biologici. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/94231
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