Since its conception, scientists have utilized the microscope to investigate the marvels of nature and biology, revealing details that would otherwise be invisible to the naked eye. Microscopes have become increasingly sophisticated and complex instruments as a result of advances in optics and photonics, and the introduction of laser illumination has fundamentally changed the concept of microscopy, shifting it from simple morphological observation to the recognition of chemical structures. The exceptional potential of nonlinear optical microscopy as a quick, label-free, highly specific, and high-resolution technology, as well as its benefits over standard methods, has been shown. In addition, various nonlinear microscopy modes may be combined in a single microscope to fully use the information capacity that multimodal pictures can give. In this thesis, after the introductory chapter and the chapter on theoretical foundations, three experimental works that I conducted during my Ph.D. are presented. These three works explore different facets of the field of nonlinear optical microscopy, giving attention to biological applications. A chapter is dedicated to each of them. First of all, I focused my research on photodamage mechanisms: given the rising usage of pulsed lasers in biophotonics applications, it is critical to ensure that the sample is not damaged by the laser while being observed. So, I studied the response of tumor cells to near-infrared ultrashort pulsed laser light. I measured the survival rate of HeLa cells as a function of laser power, scanning speed, and exposure period, irradiating them with 130-fs pulses at 1040 nm wavelength and 80 MHz repetition rate in two distinct illumination settings. To give relevant insights into safe working circumstances to many researchers planning experiments requiring laser illumination, the outcomes of the experiments were examined using a data-driven method and then confirmed with thermodynamic considerations. Then, I developed and optimized a multimodal nonlinear optical microscope able to acquire images in five different modalities: bright-field, two-photon excitation fluorescence, second harmonic generation, coherent anti-Stokes Raman scattering and stimulated Raman scattering. Using this technology, I conducted a study on murine spine section samples provided by Humanitas-Clinical and Research Centre, to investigate the role of the enzyme dipeptidyl peptidase 3 (DPP3) in bone pathology as well as the maintenance of bone homeostasis. Relevant biological changes were revealed using our multimodal microscope after highlighting discrepancies between healthy wild-type samples and DPP3 knock-out samples that had not been disclosed using standard approaches. Because of their label-free, chemically selective, and non-invasive qualities, nonlinear optical microscopy techniques have proven to be legitimate and successful in the investigation of bone diseases, as well as a valuable tool for biological research. Finally, I delved into the problem of aberrations applying sensorless adaptive optics to nonlinear microscopy. Variations in the refractive index of the sample under observation introduce optical aberrations, which degrade the quality of the image produced. It is essential that the center of mass of the system point diffusion function remains unchanged during correction, and for this to happen, the base used for correction must have the orthogonal gradient property. While it has been shown that the Lukosz Polynomials base possesses this characteristic for linear processes, no known basis has been shown to be so for multiphotonic processes. To solve this issue, I proposed an experimental approach for determining shift-less aberration bases for nonlinear microscopy applications, validating it by making in vivo images of mouse brain.
Fin dalla sua invenzione, gli scienziati hanno utilizzato il microscopio per indagare le meraviglie della natura e della biologia, rivelando dettagli che altrimenti sarebbero invisibili ad occhio nudo. I microscopi sono diventati strumenti sempre più sofisticati e complessi grazie ai progressi dell'ottica e della fotonica, e l'introduzione dell'illuminazione laser ha cambiato radicalmente il concetto di microscopia, spostandolo dalla semplice osservazione morfologica al riconoscimento delle strutture chimiche. Il potenziale eccezionale della microscopia ottica non lineare come tecnologia rapida, senza bisogno di marcatori, altamente specifica e ad alta risoluzione, così come i suoi benefici rispetto i metodi standard, sono stati ampiamente dimostrati. Inoltre, le varie modalità non lineari di microscopia possono essere combinate in un singolo microscopio per sfruttare a pieno la capacità delle informazioni che le immagini multimodali possono dare. In questa tesi, dopo il capitolo introduttivo e il capitolo sui fondamenti teorici, vengono presentati tre lavori sperimentali che ho condotto durante il mio dottorato. Questi tre lavori esplorano diversi aspetti della microscopia ottica non lineare, prestando particolare attenzione alle applicazioni biologiche. A ciascuno di essi è dedicato un capitolo. In primo luogo, ho concentrato la mia attività di ricerca sui meccanismi di fotodanneggiamento: dato l'uso crescente dei laser pulsati nelle applicazioni di biofotonica, è cruciale assicurarsi che il campione non venga danneggiato dal laser mentre viene osservato. Quindi, ho studiato la risposta delle cellule tumorali alla luce laser ultracorta nel vicino infrarosso. Ho misurato il tasso di sopravvivenza delle cellule HeLa in funzione della potenza del laser, della velocità di scansione e del tempo di esposizione, irradiandole con impulsi di 130-fs a 1040 nm di lunghezza d'onda e 80 MHz di frequenza di ripetizione in due configurazioni di illuminazione distinte. Per fornire informazioni rilevanti sulle circostanze di lavoro sicure a molti ricercatori che pianificano esperimenti che richiedono l'illuminazione laser, i risultati degli esperimenti sono stati esaminati utilizzando un metodo data-driven e quindi confermati con considerazioni termodinamiche. Poi, ho sviluppato e ottimizzato un microscopio ottico multimodale non lineare in grado di acquisire immagini in cinque diverse modalità: bright-field, fluorescenza a due fotoni, generazione di seconda armonica, coherent anti-Stokes Raman scattering e stimulated Raman scattering. Utilizzando questa tecnologia, ho condotto uno studio su campioni di sezioni di colonna vertebrale murina forniti da Humanitas-Clinical and Research Centre, per indagare il ruolo dell'enzima dipeptidil peptidase 3 (DPP3) nelle patologie ossee e nel mantenimento dell'omeostasi ossea. Facendo uso del nostro microscopio multimodale, dopo l'evidenziazione di discrepanze fra campioni sani e campioni con soppressione di DPP3, sono stati rivelati cambiamenti biologici rilevanti che non erano emersi facendo uso dei metodi standard. Grazie alle loro qualità di non necessitare di marcatori, di essere chimicamente selettive e non invasive, le tecniche di microscopia ottica non lineare hanno dimostrato di essere valide e di successo nello studio delle malattie ossee, nonché uno strumento prezioso per la ricerca biologica. Infine, ho approfondito il problema delle aberrazioni applicando l'ottica adattiva sensorless alla microscopia non lineare. Le variazioni dell'indice di rifrazione del campione sotto osservazione introducono aberrazioni ottiche che degradano la qualità dell'immagine prodotta. È essenziale che il centro di massa della point spread function del sistema rimanga invariato durante la correzione, e perché ciò avvenga, la base utilizzata per la correzione deve avere la proprietà di essere gradiente ortogonale. Mentre è stato dimostrato che la base dei polinomi di Lukosz possiede questa caratteristica per i processi lineari, non è stata dimostrata alcuna base conosciuta per i processi multiphotonici. Per risolvere questo problema, ho proposto un approccio sperimentale per la determinazione delle basi di aberrazione shift-less per le applicazioni di microscopia non lineare, convalidandolo facendo immagini in vivo di cervello di topo.
Multimodal nonlinear optical microscopy for biological applications
TALONE, BENEDETTA
2021/2022
Abstract
Since its conception, scientists have utilized the microscope to investigate the marvels of nature and biology, revealing details that would otherwise be invisible to the naked eye. Microscopes have become increasingly sophisticated and complex instruments as a result of advances in optics and photonics, and the introduction of laser illumination has fundamentally changed the concept of microscopy, shifting it from simple morphological observation to the recognition of chemical structures. The exceptional potential of nonlinear optical microscopy as a quick, label-free, highly specific, and high-resolution technology, as well as its benefits over standard methods, has been shown. In addition, various nonlinear microscopy modes may be combined in a single microscope to fully use the information capacity that multimodal pictures can give. In this thesis, after the introductory chapter and the chapter on theoretical foundations, three experimental works that I conducted during my Ph.D. are presented. These three works explore different facets of the field of nonlinear optical microscopy, giving attention to biological applications. A chapter is dedicated to each of them. First of all, I focused my research on photodamage mechanisms: given the rising usage of pulsed lasers in biophotonics applications, it is critical to ensure that the sample is not damaged by the laser while being observed. So, I studied the response of tumor cells to near-infrared ultrashort pulsed laser light. I measured the survival rate of HeLa cells as a function of laser power, scanning speed, and exposure period, irradiating them with 130-fs pulses at 1040 nm wavelength and 80 MHz repetition rate in two distinct illumination settings. To give relevant insights into safe working circumstances to many researchers planning experiments requiring laser illumination, the outcomes of the experiments were examined using a data-driven method and then confirmed with thermodynamic considerations. Then, I developed and optimized a multimodal nonlinear optical microscope able to acquire images in five different modalities: bright-field, two-photon excitation fluorescence, second harmonic generation, coherent anti-Stokes Raman scattering and stimulated Raman scattering. Using this technology, I conducted a study on murine spine section samples provided by Humanitas-Clinical and Research Centre, to investigate the role of the enzyme dipeptidyl peptidase 3 (DPP3) in bone pathology as well as the maintenance of bone homeostasis. Relevant biological changes were revealed using our multimodal microscope after highlighting discrepancies between healthy wild-type samples and DPP3 knock-out samples that had not been disclosed using standard approaches. Because of their label-free, chemically selective, and non-invasive qualities, nonlinear optical microscopy techniques have proven to be legitimate and successful in the investigation of bone diseases, as well as a valuable tool for biological research. Finally, I delved into the problem of aberrations applying sensorless adaptive optics to nonlinear microscopy. Variations in the refractive index of the sample under observation introduce optical aberrations, which degrade the quality of the image produced. It is essential that the center of mass of the system point diffusion function remains unchanged during correction, and for this to happen, the base used for correction must have the orthogonal gradient property. While it has been shown that the Lukosz Polynomials base possesses this characteristic for linear processes, no known basis has been shown to be so for multiphotonic processes. To solve this issue, I proposed an experimental approach for determining shift-less aberration bases for nonlinear microscopy applications, validating it by making in vivo images of mouse brain.| File | Dimensione | Formato | |
|---|---|---|---|
|
Talone_PhD_Thesis.pdf
accessibile in internet per tutti
Descrizione: Talone PhD thesis
Dimensione
89.7 MB
Formato
Adobe PDF
|
89.7 MB | Adobe PDF | Visualizza/Apri |
I documenti in POLITesi sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/10589/183694