The advent of nonlinear optics and its application in microscopy has opened new perspectives in the fields of biology, medicine, chemistry and physics. For example, this technique enables us to see cell details never seen before, or Raman technique, spontaneous and coherent, allows us to distinguish chemical components of matter from its spectrum. All these achievements are possible thanks to pulsed laser sources. Particularly interesting is the possibility of doing in vivo measurements and imaging on biological cells and in this case speed is the key factor. It is necessary to have a very high signal-to-noise ratio (SNR) in order to make this technology accesible produce images at video rate. A high signal-to-noise ratio can be obtained by increasing the power of the pumping laser, but care must be taken not to damage the sample being examined. With biological samples, coherent Raman microscopy (Stimulated Raman Scattering, SRS, and Coherent Anti-Stokes Raman Scattering, CARS) can be performed using IR laser pulses that are not absorbed by the cells. Linear absorption is not the only damaging factor in which one can incur using pulsed laser sources, and while looking for best possible signal-to-noise ratio, other phenomena can be generated, i.e. multiphoton absorption and thermal and chemical effects, that damage the sample and compromise the result of measurements. During this thesis project a study was carried out about the damage induced by an infrared pulsed laser source on cellular samples. As starting point, we decided to use a laser source at 1040nm because this project is part of the work of a research group, in which the aim is to realize Raman SRS microscopy using a pump laser at wavelength and a broadband laser in the infrared for the Stokes laser. Among the various cell lines, we choose HeLa cells and the measurements were carried out by systematically varying the laser power, the pulse width and the scanning speed. In this way, we succeded in defining the damage threshold in a 3-variables space and in assessing that damage is mainly caused by multiphotonic processes.
L'avvento dell'ottica non lineare e la sua applicazione in microscopia ha aperto nuove strade nei campi della biologia, medicina, chimica e fisica. Tutto ciò è reso possibile dall'utilizzo di sorgenti laser impulsate. Grande interesse desta anche la possibilità di fare misure e imaging su cellule e tessuti biologici in vivo, e per questo motivo la velocità diventa un fattore determinante. Per rendere fruibile tale tecnologia e produrre immagini a video rate è necessario avere un rapporto segnale/rumore (SNR) molto alto. Questa condizione è ottenibile aumentando la potenza del laser di pompa, ma bisogna prestare molta attenzione a non danneggiare il campione che si sta esaminando. Quando si utilizzano campioni biologici, è possibile fare microscopia Raman coerente (Stimulated Raman Scattering, SRS, e Coherent Anti-Stokes Raman Scattering, CARS) utilizzando impulsi laser nell'infrarosso che non vengono assorbite dalle cellule. L'assorbimento lineare non è l'unico fattore di danneggiamento in cui si può incorrere utilizzando sorgenti laser impulsate, e nella ricerca del miglior SNR possibile, si rischia di generare altri fenomeni, quali assorbimento multifotonico ed effetti termici e chimici. che possono compromettere il risultato della misura. In questo progetto di tesi è stato condotto uno studio del danneggiamento indotto su campioni cellulari esposti ad una sorgente laser impulsata infrarossa. Come punto di partenza si è scelto di utilizzare una sorgente di lunghezza d'onda 1040nm perchè questo progetto rientra nel lavoro di un gruppo di ricerca in cui si sta lavorando per realizzare microscopia Raman SRS utilizzando un laser di pompa a 1000nm di lunghezza d'onda e un laser a banda larga nell'infrarosso per il laser Stokes. Come linea cellulare sono state utilizzate cellule HeLa e le misure sono state svolte variando in modo sistematico la potenza del laser, la larghezza dell'impulso e la velocità di scansione. Siamo stati così in grado di definire la soglia di danneggiamento in uno spazio a tre variabili stimando che con queste lunghezze d'onda il danneggiamento avvenga per processi multifotonici.
Biological damage induced in living cells by focused ultrashort laser pulses
TALONE, BENEDETTA
2017/2018
Abstract
The advent of nonlinear optics and its application in microscopy has opened new perspectives in the fields of biology, medicine, chemistry and physics. For example, this technique enables us to see cell details never seen before, or Raman technique, spontaneous and coherent, allows us to distinguish chemical components of matter from its spectrum. All these achievements are possible thanks to pulsed laser sources. Particularly interesting is the possibility of doing in vivo measurements and imaging on biological cells and in this case speed is the key factor. It is necessary to have a very high signal-to-noise ratio (SNR) in order to make this technology accesible produce images at video rate. A high signal-to-noise ratio can be obtained by increasing the power of the pumping laser, but care must be taken not to damage the sample being examined. With biological samples, coherent Raman microscopy (Stimulated Raman Scattering, SRS, and Coherent Anti-Stokes Raman Scattering, CARS) can be performed using IR laser pulses that are not absorbed by the cells. Linear absorption is not the only damaging factor in which one can incur using pulsed laser sources, and while looking for best possible signal-to-noise ratio, other phenomena can be generated, i.e. multiphoton absorption and thermal and chemical effects, that damage the sample and compromise the result of measurements. During this thesis project a study was carried out about the damage induced by an infrared pulsed laser source on cellular samples. As starting point, we decided to use a laser source at 1040nm because this project is part of the work of a research group, in which the aim is to realize Raman SRS microscopy using a pump laser at wavelength and a broadband laser in the infrared for the Stokes laser. Among the various cell lines, we choose HeLa cells and the measurements were carried out by systematically varying the laser power, the pulse width and the scanning speed. In this way, we succeded in defining the damage threshold in a 3-variables space and in assessing that damage is mainly caused by multiphotonic processes.| File | Dimensione | Formato | |
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https://hdl.handle.net/10589/139092