Broadband time-domain diffuse optics for clinical diagnostics, and diffuse Raman spectroscopy

The mystery of light-tissue interaction explored by the scientific evolution can be classified into few set of basic phenomena like absorption of photons, Raman effects, fluorescence effect, stimulated emission, two photon absorption, etc. These inventions played significant role in enhancing the specificity of molecular detection, thereby revolutionizing the field of in vitro tissue diagnostics and microscopy. However, the understanding of scattering in tissues is still at its evolutionary stage where steadfast attempts have been made by the scientific community to demystify the secrets of light propagation in diffusive media like human tissues. In the recent decade, diffuse optics has been successful in advancing the studies on light-tissue interaction in diffusive media. This PhD thesis stands at this juncture of scientific revolution, contributing to the invention and revelation of advance techniques to understand the interaction and propagation of photons in diffusive media and there by propelling the next-gen non-invasive diagnosis of biological tissues. The research leading to this PhD dissertation has been carried out in the physics department of Politecnico di Milano (Milan, Italy), and two secondments at The Institute of Photonic Sciences (ICFO - Barcelona, Spain), in collaboration with other organizations (PicoQuant - Berlin,Germany) and hospitals(Hospital Universitari Germans Trias i Pujol - Badalona, and HospitalClinic Barcelona - Barcelona,Spain) The essence of the thesis can be broken into two parts: 1. Time-domain Diffuse Optical Spectroscopy (TD-DOS) for Clinical Diagnostics. 2. Diffuse Raman Spectroscopy (DRS). The key achievements of thesis are summarized below: Though diffuse optical studies are possible by continuous wave (CW) or modulated light sources. Time-domain diffuse optics is an advanced tool among them, as it can naturally disentangle absorption from scattering, providing high spatial and depth resolution. The entire DOS part of the thesis V was carried out with time-domain techniques. Chapter 1 serves as an introduction and provides preliminary basis for the rest of the thesis. Various theoretical models of diffusion exploited by this thesis for data analysis are elaborated on chapter 2. Chapter:3 Development of broadband (500-1350 nm) TD-DOS spectrometer Typically, broadband TD-DOS on human tissues are carried out over 600-1000 nm. Though the tissue spectrum has interesting features beyond 1100 nm, the absence of broadband detectors coupled with stringent requirements of time-domain optics and theoretical modelling demand a generous spectral extension. During this thesis, a successful extension was achieved by adapting key strategies on source and detection chain optics, fibers, responsivity extension with two detectors and drift and distortion compensation by IRF acquisition. Additionally, this system was designed and validated in the view of clinical application, thus making it a unique masterpiece and an invaluable instrument in the field of TD-DOS. Chapter:4 Discovery and characterization of new tissue constituents Four tissue constituents namely: collagen, elastin, tyrosine and thyroglobulin were characterized during this thesis period. Moreover, high scattering, fluorescence, boundary effect and bandwidth effects were some of the problems tackled in this work by means of customized probes, Monte Carlo models, bandwidth simulations and fluorescence filters, to extract absorption spectra. The characterized collagen and elastin spectra (500-1700 nm) can be key to new exploration in the beyond range (>1100 nm); breast tissue diagnosis, elasticity assessment being a few potential areas of application. Tyrosine and Thyroglobulin are tissue constituents specific to thyroid organ, and the work done on these constituents has direct impact on non-invasive diagnosis of thyroid pathologies. Chapter:5 Broadband in vivo studies on: Human bone, Thyroid, and Abdomen The broadband clinical system developed during this thesis was employed in multiple hospitals. A well deliberated protocol was carried out to measure non-invasively the above-mentioned locations. The presence of collagen in bone tissue revealed by our studies emphasized its relevance to the global diagnosis of bone pathologies like osteoporosis. The high absorption of thyroid organ led us to the discovery of new tissue constituents (thyroglobulin, tyrosine) that were absent in our data analysis, thus unlocked the new methods in thyroid diagnosis. The preliminary results of the abdomen studies for fat distinction, revealed its potential to distinguish multi-layered structure of abdomen tissues. Chapter:6 Invention of Frequency Offset Raman Spectroscopy for deep tissue Raman Spectroscopy Raman spectroscopy of diffusive media has been explored in the recent decade to extract Raman spectrum of deep tissues. The method based on multidistance approach, Spatial Offset Raman Spectroscopy (SORS) has revolutioned the CW diffuse Raman spectroscopy. In this dissertation, an alternative technique, Frequency Offset Raman Spectroscopy (FORS) to probe deep into tissue was proposed and demonstrated on tissue mimicking solid phantoms. The proposed technique is performed at multiple excitation wavelengths and utilizes variations in optical properties (absorption, scattering) to probe deep into tissues. The figure depicts the Raman probe and the principle of FORS. FORS has been found to have superior spatial resolution and low signal to noise ratio as compared to SORS thereby making it as a valid alternative to the existing SORS. VI Contents Chapter:7 Time-domain Diffuse Raman Spectroscopy (TD-DRS) The aim of this work was to transfer the know-how developed in the last 20 years of Time-Domain Diffuse Optics (TD-DO) to the realm of Raman spectroscopy and to lay a strong foundation by the theoretical and experimental realization of TD-DRS. A new analytical model in collaboration with the University of Florence was developed to answer the propagation of Raman photons in diffusive media. Unfortunately, the experimental realization of diffuse Raman is complex due to very low signal (factor of 10-9 as compared to Tyndall) scattering. However, a customized spectrometer and probe were built to enable the sequential detection of time resolved Raman photons thereby opening up new horizons in the branch of TDDRS. Furthermore, a new detector technology developed by PicoQuant was exploited to make parallel acquisition of TD-Raman curves. Preliminary results revealed feasibility of the TD-DRS measurement with single-photon counting and demonstrated depth sensitivity of the approach.

Il mistero dell'interazione luce-tessuti studiato dall'evoluzione scientifica può essere classificato in pochi fenomeni base come l'assorbimento di fotoni, gli effetti Raman, la fluorescenza, l'emissione stimolata, l'assorbimento a due fotoni, ecc. Questi fenomeni hanno svolto un ruolo significativo nel migliorare la specificità della rivelazione molecolare, rivoluzionando il campo della diagnostica dei tessuti "in vitro" e la microscopia. Nell'ultima decade, l'ottica diffusa ha avuto successo nel progredire negli studi sull'interazione luce-tessuti nei mezzi diffondenti. Questa tesi di dottorato si colloca a questo punto della rivoluzione scientifica, contribuendo all'invenzione e rivelazione di tecniche avanzate per comprendere l'interazione e la propagazione di fotoni in mezzi diffondenti, quindi spingendo verso la prossima generazione di diagnosi non invasiva di tessuti biologici. Sebbene gli studi di ottica diffusa siano possibili in continua (Continuous Wave - CW) o con sorgenti di luce modulate, tra questi l'ottica diffusa nel dominio del tempo è uno strumento più avanzato, poichè può naturalmente disaccoppiare l'assorbimento dalla diffusione, fornendo elevata risuluzione spaziale e in profondità. L'intera parte DOS (Diffuse Optical Spectroscopy) della tesi è stata condotta con tecniche nel dominio del tempo. Nel mio lavoro, mi sono concentrato sullo sviluppo di uno spettrometro a banda estesa (500-1350 nm), adottando strategie chiave sulla sorgente e la catena ottica di rivelazione, fibre, estensione della responsibità con due rivelatori e compensazione di deriva e distorsione tramite l'acquisizione della funzione di risposta all'impulso (Impulse Response Function - IRF). Inoltre, questo sistema è stato progettato e validato in vista dell'applicazione clinica, rendendolo quindi un strumento unico e di valore inestimabile nel campo della Spettroscopia Ottica Diffusa nel dominio del tempo (Time Domain Diffuse Optical Spectroscopy). In seguito, sono stati caratterizzati alcuni costituenti dei tessuti come collagene, elastina, tirosina e tireoglobulina. Ho condotto studi "in vivo" su ampia banda spettrale su ossa umane, tiroide e addome, identificando i loro principali costituenti, cosa estremamente rilevante per la diagnostica clinica. La seconda parte del mio lavoro si è focalizzata sulla Spettroscopia Raman dei tessuti profondi. La Spettroscopia Raman dei mezzi diffondenti è stata esplorata nell'ultima decade per estrarre lo spettro Raman dei tessuti profondi. Il metodo basato su un approccio multi-distanza, la Spettroscopia Raman con Offset Spaziale (Spatial Offset Raman Spectroscopy - SORS) ha rivoluzionato la spettroscopia Raman in continua. In questa dissertazione, viene proposta e testata, su phantoms che modellizzano i tessuti solidi, una tecnica alternativa per sondare i tessuti in profondità: la Spettroscopia Raman con Offset in Frequenza (Frequency Offset Raman Spectroscopy - FORS). L'obiettivo di questa parte del mio lavoro è stato trasferire il know-how sviluppato negli ultimi vent'anni nell'Ottica Diffusa nel Dominio del Tempo (Time Domain Diffuse Optics - TD-DO) alla realtà della Spettroscopia Raman, mettendo delle solide basi per la realizzazione teorica e sperimentale della Spettroscopia Raman Diffusa nel Dominio del Tempo (Time Domain Diffuse Raman Spectroscopy - TD DRS). I risultati preliminari hanno rivelato la fattibilità delle misure TD-DRS in regime di conteggio di singolo fotone e mostrato la sensibilità alla profondità di tale tecnica.