Label-free three-dimensional imaging of Caenorhabditis elegans with visible optical coherence microscopy

Fast, label-free, high-resolution, three-dimensional imaging platforms are crucial for high-throughput in vivo time-lapse studies of the anatomy of Caenorhabditis elegans, one of the most commonly used model organisms in biomedical research. Despite the needs, methods combining all these characteristics have been lacking. Here, we present label-free imaging of live Caenorhabditis elegans with three-dimensional sub-micrometer resolution using visible optical coherence microscopy (visOCM). visOCM is a versatile optical imaging method which we introduced recently for tomography of cell cultures and tissue samples. Our method is based on Fourier domain optical coherence tomography, an interferometric technique that provides three-dimensional images with high sensitivity, high acquisition rate and micrometer-scale resolution. By operating in the visible wavelength range and using a high NA objective, visOCM attains lateral and axial resolutions below 1 μm. Additionally, we use a Bessel illumination offering an extended depth of field of approximately 40 μm.We demonstrate that visOCM’s imaging properties allow rapid imaging of full sized living Caenorhabditis elegans down to the sub-cellular level. Our system opens the door to many applications such as the study of phenotypic changes related to developmental or ageing processes.

An automated microfluidic platform for C. elegans embryo arraying, phenotyping, and long-term live imaging

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Virginija Jovaisaite, Thomas Lehnert, Alexis Pierre Henri Marette, Laurent Mouchiroud, Shreya Narasimhan

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population's statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPR(mt)) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Nature Publishing Group2015

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography

Arno Pino Bouwens, Séverine Coquoz, Adrien Charles-François Raymond Descloux, Jérôme Extermann, Theo Lasser, Paul James Marchand, David Nguyen, Miguel Sison, Daniel Pawel Szlag

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 μm axial and 0.4 μm lateral resolution maintained over a depth of 40 μm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.

Optical Soc Amer2017

Coherent imaging from bacteria to multicellular organisms

Séverine Coquoz

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.

EPFL2017

Coherent imaging from bacteria to multicellular organisms

Séverine Coquoz

EPFL2017