Phase contrast (PC) microscopy offers notable opportunities for optical label-free studies in cellular biology. Indeed, the phase of light travelling through a sample provides a contrast mechanism, which codes its thickness (morphology) or refractive index (biochemistry). As such, PC methods allow imaging transparent biological samples without causing photobleaching and phototoxicity as in fluorescence microscopy. However, quantitative phase contrast methods, e.g., differential interference contrast microscopy, are based on interferometry. This architecture is sensitive to misalignments between the probe and reference beams, external disturbances, and its integration into other microscopes can be complicated and costly. A method capable of solving the above problems is ptychography, namely a computational microscopy technique that computes the phase and amplitude of light waves probing a target sample by analyzing multiple diffraction patterns. Compared to other PC methods, ptychography has unique features: 1) the experimental setup can be as simple as a light source and camera; as such, the technique can be integrated into a large variety of microscopes with minimal additional optics; 2) Ptychography applies to thin and thick samples, as well; 3) returning both amplitude and phase images, ptychography is inherently a multimodal system. This thesis aims at exploiting optical ptychography as a label-free tool for biological studies. To this end, we designed and characterized a compact ptychography microscope, which can be easily integrated into other microscopes leading to a multimodal imaging system. We found that our ptychographic system has excellent phase contrast sensitivity (5.34×106 rad/m∙RIU) and low detection limit (8.8×10-7 m∙RIU). Following this result, we used ptychography to study genetic diseases. As a case of study, we selected the Hutchinson-Gilford progeria syndrome based on the rationale that healthy, and progeria cells have different chromatin compactions and thus different refractive indices. As our results indicate, ptychography measured notable differences between the phase of healthy and unhealthy cells, which were confirmed by multimodal measurements comprising confocal and light-polarized microscopy. Although we are aware that there is still room for improvements– the spatial resolution of our system is a point requiring future works– this thesis undoubtedly is a significant step forward in the realization of a totally label-free multimodal microscope for advanced biological studies.

DEVELOPING OPTICAL PTYCHOGRAPHY FOR BIOPHYSICAL APPLICATIONS

TRIANNI, ALBERTA
2022-06-21

Abstract

Phase contrast (PC) microscopy offers notable opportunities for optical label-free studies in cellular biology. Indeed, the phase of light travelling through a sample provides a contrast mechanism, which codes its thickness (morphology) or refractive index (biochemistry). As such, PC methods allow imaging transparent biological samples without causing photobleaching and phototoxicity as in fluorescence microscopy. However, quantitative phase contrast methods, e.g., differential interference contrast microscopy, are based on interferometry. This architecture is sensitive to misalignments between the probe and reference beams, external disturbances, and its integration into other microscopes can be complicated and costly. A method capable of solving the above problems is ptychography, namely a computational microscopy technique that computes the phase and amplitude of light waves probing a target sample by analyzing multiple diffraction patterns. Compared to other PC methods, ptychography has unique features: 1) the experimental setup can be as simple as a light source and camera; as such, the technique can be integrated into a large variety of microscopes with minimal additional optics; 2) Ptychography applies to thin and thick samples, as well; 3) returning both amplitude and phase images, ptychography is inherently a multimodal system. This thesis aims at exploiting optical ptychography as a label-free tool for biological studies. To this end, we designed and characterized a compact ptychography microscope, which can be easily integrated into other microscopes leading to a multimodal imaging system. We found that our ptychographic system has excellent phase contrast sensitivity (5.34×106 rad/m∙RIU) and low detection limit (8.8×10-7 m∙RIU). Following this result, we used ptychography to study genetic diseases. As a case of study, we selected the Hutchinson-Gilford progeria syndrome based on the rationale that healthy, and progeria cells have different chromatin compactions and thus different refractive indices. As our results indicate, ptychography measured notable differences between the phase of healthy and unhealthy cells, which were confirmed by multimodal measurements comprising confocal and light-polarized microscopy. Although we are aware that there is still room for improvements– the spatial resolution of our system is a point requiring future works– this thesis undoubtedly is a significant step forward in the realization of a totally label-free multimodal microscope for advanced biological studies.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11567/1087784
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