Single-cell analysis has become an important field of research in biology in the last decade. The study of the characteristics and of the behaviour of single cells helped the biologists to understand the effect of heterogeneity in cellular populations and deepened their knowledge in many physiological and pathological processes, one above all cancer progression. New techniques have been developed in order to analyze cells one by one. Microfluidics, in particular, offers the possibility to address single cells with specific stimuli and to analyze their response or characteristics, guaranteeing at the same time a high throughput. A particular technique, called optofluidics, consists in integrating optic based techniques for cellular inspection and microfluidic platforms for sample handling. During my work, I developed and characterized four different optpofluidic micro-devices for single-cell analysis. The first device integrates a particular microscopy technique, called light-sheet fluorescence microscopy, into a microfluidic platform, in order to study heterogeneity of cancer population at single-cell level. A second device consists in the integration of light-sheet based structured illumination microscopy in the same microfluidic platform of the first device, in order to push the system resolution over the standard optical diffraction limit. The third device is a microfluidic platform that allows the study and the imaging of single-cells migration strategies in a high-confining extracellular matrix. The fourth device is an optofluidic platform for unicellular algae study and it will act as a core for an automated setup for in-situ microalgae population screening. All the chips have been validated with biological samples. All these devices have been realized with a direct-writing laser technique called femtosecond laser micromachining. This technique can microstructure different materials, such as glass and polymeric resits, and it allows the realization of photonic devices, microfluidic networks and embedded micro-optics on the same substrate. During my work I fully exploited the capability of this technique to combine optical and microfludic components in the same device, realizing three-dimensional and integrated optofluidic lab-on-chips with micrometric features size.
Nell’ultimo decennio, l’analisi single-cell è diventato un importante campo di ricerca per la biologia. Lo studio delle caratteristiche e del comportamento delle singole cellule ha aiutato gli scienziati a comprendere l’effetto dell’eterogeneità nelle popolazioni cellulari e ha approfondito la loro comprensione di molti processi fisiologici e patologici, uno su tutti l’evoluzione del cancro. Diverse nuove tecniche sono state sviluppate per poter analizzare le cellule una ad una. La microfluidica, in particolare, offre la possibilità di applicare stimoli specifici alle singole cellule ed analizzare la loro risposta o le loro caratteristiche, garantendo al contempo un elevato numero di acquisizioni al minuto. Una particolare tecnica, chiamata optofluidica, consiste nell’integrazione di tecniche ottiche per l’analisi cellulare e di piattaforme microfluidiche per la manipolazione di campioni Nel mio lavoro, ho sviluppato e caratterizzato quattro diversi micro-dispositivi optofluidici per l’analisi single-cell. Un primo dispositivo integra una particolare tecnica di microscopia, chiamata light-sheet fluorescence microscopy, in una piattaforma microfluidica, in modo da poter investigare l’eterogeneità di una popolazione cancerogena al livello di singole cellule. Un secondo dispositivo consiste nell’integrazione della tecnica structured illumination microscopy basata su foglietto di luce nella stessa piattaforma fluidica del primo dispositivo, in modo da spingere la risoluzione del Sistema oltre il limite di diffrazione ottico. Il terzo dispositivo è una piattaforma fluidica che permette lo studio e l’imaging di singole cellule durante la loro migrazione in una matrice extracellualre rigida ad alto confinamento. Il quarto dispositivo è una piattaforma optofluidica per lo studio di alghe unicellulare e che sarà il cuore di una sistema automatizzato per il monitoraggio in-situ della popolazione di microalghe del lago di Ginevra. Tutti questi microchip sono stati validati con campioni biologici. Tutti i dispositivi sono stati realizzati tramite una tecnica di scrittuara laser diretta chiamata femtosecond laser micromachining. Questa tecnilogia permette di microstrutturare diversi materiali, come il vetro o delle resine pomileriche e può essere utilizzata per la realizzazione di dispositivi fotonici, circuiti microfluidici e micro-ottiche integrate, il tutto sullo stesso substrato. Durante il mio lavoro ho sfruttato appieno le potenzialità di questa tecnica per combinare componenti ottici e microfluidici nello stesso dispositivo, realizzando Lab-On-Chip optofluidici integrati ed altamente treidimensionali , con dimensioni micrometriche
Optofluidic biochips for single-cells imaging fabricated with femtosecond laser micromachining
Sala, Federico
2021/2022
Abstract
Single-cell analysis has become an important field of research in biology in the last decade. The study of the characteristics and of the behaviour of single cells helped the biologists to understand the effect of heterogeneity in cellular populations and deepened their knowledge in many physiological and pathological processes, one above all cancer progression. New techniques have been developed in order to analyze cells one by one. Microfluidics, in particular, offers the possibility to address single cells with specific stimuli and to analyze their response or characteristics, guaranteeing at the same time a high throughput. A particular technique, called optofluidics, consists in integrating optic based techniques for cellular inspection and microfluidic platforms for sample handling. During my work, I developed and characterized four different optpofluidic micro-devices for single-cell analysis. The first device integrates a particular microscopy technique, called light-sheet fluorescence microscopy, into a microfluidic platform, in order to study heterogeneity of cancer population at single-cell level. A second device consists in the integration of light-sheet based structured illumination microscopy in the same microfluidic platform of the first device, in order to push the system resolution over the standard optical diffraction limit. The third device is a microfluidic platform that allows the study and the imaging of single-cells migration strategies in a high-confining extracellular matrix. The fourth device is an optofluidic platform for unicellular algae study and it will act as a core for an automated setup for in-situ microalgae population screening. All the chips have been validated with biological samples. All these devices have been realized with a direct-writing laser technique called femtosecond laser micromachining. This technique can microstructure different materials, such as glass and polymeric resits, and it allows the realization of photonic devices, microfluidic networks and embedded micro-optics on the same substrate. During my work I fully exploited the capability of this technique to combine optical and microfludic components in the same device, realizing three-dimensional and integrated optofluidic lab-on-chips with micrometric features size.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/183699