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doctoral thesis

Coherent imaging from bacteria to multicellular organisms

Coquoz, Séverine  
2017

Structural and functional imaging of cells, tissues and organisms is crucial for understanding biomedical processes. Fluorescence microscopy is an established tool and has contributed to many discoveries in the life sciences. This technique provides molecular contrast but has limitations inherent to the fluorescent probes, namely phototoxicity and photobleaching. Phase microscopy provides a label-free alternative. Contrast is achieved by translating the phase variations induced by the sample into measurable intensity variations. While phase contrast and differential interference contrast microscopy offer a means to generate contrast without exogenous contrast agents, quantitative phase imaging (QPI) techniques have emerged, enabling the measurement of the phase delays. This measurement yields information on the thickness, refractive index and dry mass of the sample. QPI is thus a powerful tool and has proven its use in various biomedical applications. In the first part of this thesis, new concepts for extending the capabilities of QPI and applications exploiting them are presented. We implement piezo-based Fourier phase microscopy, exhibiting high spatial and temporal sensitivities and resolutions for label-free imaging of cell dynamics at various time scales. The phase is retrieved via phase-shifting intereferometry using a piezo-actuated module allowing very fast tunable phase modulation. Several examples of applications are described. Furthermore, we exploit the phase-to-mass equivalence for investigating the growth of uropathogenic E. coli and the effect of different antibiotics, thereby providing new insights into fundamental mechanisms of bacterial growth. Because QPI methods are limited to optically thin and weakly scattering objects, the second part of this thesis focuses on optical coherence microscopy (OCM). OCM is an interferometric technique providing 3D images of highly scattering biological samples with micrometric resolution and penetration depth of up to several hundreds of micrometers. Compared to optical coherence tomography, OCM uses high numerical aperture (NA) optics to achieve higher transverse resolution, leading to a decrease of the depth of field (DOF). Several methods have been developed to overcome this trade-off between resolution and DOF. Extended-focus OCM (xfOCM) provides a particularly interesting solution by illuminating the sample with a Bessel beam. Moreover, interferometric synthetic aperture microscopy (ISAM) achieves depth-independent resolution through an approximate solution to the inverse scattering problem. We develop extended ISAM (xISAM) to combine the benefits of both approaches, and demonstrate its potential on simulated and experimental data. We also propose a new application of visible OCM (visOCM), a xfOCM system using visible light and a high-NA objective to achieve 3D imaging with sub-micrometer spatial resolution. The capabilities of visOCM are well suited for 3D in vivo imaging of C. elegans, an extensively used model organism in biomedical research. We demonstrate that the anatomy of C. elegans can be visualized with high speed and high contrast down to the sub-cellular level. We further show that visOCM can be employed for imaging age-related morphological changes. Altogether, the methods developed in this thesis pave the way to many applications in the life sciences by featuring label-free imaging at various temporal and spatial scales. Several promising applications are pursued.

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Type
doctoral thesis
DOI
10.5075/epfl-thesis-7857
Author(s)
Coquoz, Séverine  
Advisors
Lasser, Theo  
•
Leutenegger, Marcel  
Jury

Prof. Hans Peter Herzig (président) ; Prof. Theo Lasser, Dr Marcel Leutenegger (directeurs) ; Prof. John McKinney, Dr Michael Kempe, Prof. Rainer Leitgeb (rapporteurs)

Date Issued

2017

Publisher

EPFL

Publisher place

Lausanne

Public defense year

2017-09-15

Thesis number

7857

Total of pages

121

Subjects

Coherent imaging

•

quantitative phase microscopy (QPM)

•

quantitative phase imaging (QPI)

•

piezo-based Fourier phase microscopy (pFPM)

•

optical coherence microscopy (OCM)

•

extended-focus (xf)

•

extended interferometric synthetic aperture microscopy (xISAM)

•

coherent transfer function (CTF)

•

bacterial growth

•

3D morphological imaging

EPFL units
LOB  
Faculty
STI  
School
IMT  
Doctoral School
EDPO  
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/140001
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