Spontaneous Raman (SR) spectroscopy is a powerful label-free optical spectroscopy tool to measure vibrational spectra of molecules, providing a unique signature exploitable for the identification of the different chemical species in heterogeneous samples. SR allows one to acquire the full spectrum covering the whole Raman active region, shining the sample with a quasi-monochromatic laser beam. However, SR suffers from low scattering cross-sections, preventing high acquisition speeds. Coherent anti-Stokes Raman scattering (CARS) overcomes this limitation, as it exploits third-order non-linear optical processes that coherently excite the vibrational modes of the investigated sample. The narrowband CARS signal derives from the interaction of the sample with two spatially and temporally synchronized picosecond pulses, namely pump and Stokes, and detects a specific vibrational mode blue-shifted with respect to the generating beams. Broadband CARS (B-CARS) combines a narrowband pump beam with a broadband Stokes beam, probing multiple vibrational transitions at the same time, detected with a spectrometer. This thesis presents an innovative B-CARS set-up, based on a fiber laser system that we use to generate a narrowband pump beam and a broadband Stokes beam via supercontinuum (SC) generation in bulk media. The work is organized as follows: Chapter 1 introduces CARS processes; Chapter 2 gives a theoretical derivation of the equations involved in CARS processes and SC generation. Chapter 3 describes the B-CARS set-up and the experimental results that I obtained during my thesis and the post-processing algorithms used to extrapolate relevant chemical information. We demonstrated that our set-up allows us to perform ultrabroadband CARS microscopy and spectroscopy, through two and three-color CARS, covering the whole Raman active region (500-3100 cm-1). We performed high-speed spectroscopy (<1 ms/spectrum) on solvents, subcellular acids and solid-state crystals in excellent agreement with SR spectra. We then validated our system imaging plastic beads and biological samples collecting hyperspectral data at high acquisition speed in a raster-scanning fashion. Through data processing, we demonstrated that our system enables us to derive concentration maps highlighting the different chemical species in unlabeled samples.
L'effetto Raman spontaneo (SR) è un potente strumento di spettroscopia ottica label-free che permette di misurare gli interi spettri vibrazionali delle molecole illuminando il campione con un fascio laser quasi-monocromatico. Questi spettri possono essere associati in modo univoco alle specie chimiche in campioni eterogenei, permettendone l'identificazione. Tuttavia, SR soffre di basse sezioni d'urto di scattering, che impediscono il raggiungimento di velocità di acquisizione elevate. Il Coherent anti-Stokes Raman scattering (CARS) supera questo limite, poiché sfrutta processi ottici non lineari del terzo ordine che eccitano coerentemente i modi vibrazionali del campione studiato. Il segnale CARS a banda stretta deriva dall'interazione del campione con due impulsi, detti pompa e Stokes, con durata nell'ordine dei picosecondi, sincronizzati spazialmente e temporalmente, e rileva uno specifico modo vibrazionale. Il segnale risultante ha una lunghezza d'onda minore dei fasci incidenti. Il CARS a banda larga (B-CARS) combina un fascio di pompa a banda stretta con un fascio di Stokes a banda larga, sondando più transizioni vibrazionali contemporaneamente, e viene rilevato con uno spettrometro. Questa tesi presenta un set-up B-CARS innovativo, basato su un sistema laser in fibra, utilizzato per generare un fascio di pompa a banda stretta e un fascio di Stokes a banda larga attraverso la generazione di luce supercontinua (SC) in un cristallo. Il lavoro è organizzato come segue: il Capitolo 1 introduce i processi CARS; il Capitolo 2 fornisce una derivazione teorica delle equazioni coinvolte nei processi CARS e nella generazione di SC. Il Capitolo 3 descrive il set-up B-CARS e i risultati sperimentali ottenuti durante la tesi, nonché gli algoritmi di post-processing utilizzati per estrapolare le informazioni chimiche. Abbiamo dimostrato che il nostro set-up permette di eseguire microscopia e spettroscopia CARS a banda ultra larga, attraverso two e three-color CARS, coprendo l'intera regione Raman (500-3100 cm-1). Abbiamo effettuato misure di spettroscopia ad alta velocità (<1 ms/spettro) su solventi, acidi subcellulari e cristalli, in ottimo accordo con gli spettri ottenuti tramite SR. Abbiamo poi eseguito l'imaging di sfere di plastica e campioni biologici, acquisendo dati iperspettrali ad alta velocità. A seguito dell'elaborazione dei dati, abbiamo dimostrato che il nostro set-up consente di ricavare mappe di concentrazione che evidenziano le diverse specie chimiche in campioni privi di labelling.
High-speed ultrabroadband CARS microscopy based on supercontinuum generation in bulk media
GUCCI, FRANCESCO
2021/2022
Abstract
Spontaneous Raman (SR) spectroscopy is a powerful label-free optical spectroscopy tool to measure vibrational spectra of molecules, providing a unique signature exploitable for the identification of the different chemical species in heterogeneous samples. SR allows one to acquire the full spectrum covering the whole Raman active region, shining the sample with a quasi-monochromatic laser beam. However, SR suffers from low scattering cross-sections, preventing high acquisition speeds. Coherent anti-Stokes Raman scattering (CARS) overcomes this limitation, as it exploits third-order non-linear optical processes that coherently excite the vibrational modes of the investigated sample. The narrowband CARS signal derives from the interaction of the sample with two spatially and temporally synchronized picosecond pulses, namely pump and Stokes, and detects a specific vibrational mode blue-shifted with respect to the generating beams. Broadband CARS (B-CARS) combines a narrowband pump beam with a broadband Stokes beam, probing multiple vibrational transitions at the same time, detected with a spectrometer. This thesis presents an innovative B-CARS set-up, based on a fiber laser system that we use to generate a narrowband pump beam and a broadband Stokes beam via supercontinuum (SC) generation in bulk media. The work is organized as follows: Chapter 1 introduces CARS processes; Chapter 2 gives a theoretical derivation of the equations involved in CARS processes and SC generation. Chapter 3 describes the B-CARS set-up and the experimental results that I obtained during my thesis and the post-processing algorithms used to extrapolate relevant chemical information. We demonstrated that our set-up allows us to perform ultrabroadband CARS microscopy and spectroscopy, through two and three-color CARS, covering the whole Raman active region (500-3100 cm-1). We performed high-speed spectroscopy (<1 ms/spectrum) on solvents, subcellular acids and solid-state crystals in excellent agreement with SR spectra. We then validated our system imaging plastic beads and biological samples collecting hyperspectral data at high acquisition speed in a raster-scanning fashion. Through data processing, we demonstrated that our system enables us to derive concentration maps highlighting the different chemical species in unlabeled samples.File | Dimensione | Formato | |
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Thesis_Francesco_Gucci.pdf
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https://hdl.handle.net/10589/195439