Coherent imaging from bacteria to multicellular organisms

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.

Functional and Structural Imaging with Optical Coherence Microscopy

Arno Pino Bouwens

Fluorescence microscopy techniques are well established research tools and have proven their use in a large variety of biomedical applications. Microscopic molecular contrast is achieved by imaging fluorescent dyes that bind specifically to a molecule of interest and gene expression can be imaged by genetically modifying organisms to express fluorescent proteins. This versatile technique has, however, three limitations: fluorescent labels can cause toxicity or have an unwanted influence on the process under study; the photobleaching effect limits observation time; and the highest acquisition speed is restricted by the fluorophore’s brightness and by the raster scan required for 3-D imaging. For applications where direct molecular contrast is not essential, label-free imaging offers a solution to these issues and can simplify imaging experiments. In this work, we present methods to perform 3-D label-free imaging of the anatomy (structural imaging) and physiology (functional imaging) of living tissue and cells with optical coherence microscopy (OCM). OCM is founded on low-coherence interferometry and acquires 3-D images of the local light back-scattering strength from within highly scattering biological tissue. By measuring in the spectral or Fourier domain, image acquisition is multiplexed over depth and achieves high sensitivity. OCM can therefore offer an important gain in acquisition speed, under the condition that lateral resolution is maintained over the imaging depth. This is realized by extended focus OCM (xfOCM), which uses Bessel beam illumination to create a high lateral resolution over an extended depth of field. The combination of high 3-D resolution and fast acquisition is especially useful for in vivo experiments, where measurement time is limited, and when fast processes are studied. We demonstrate long-term in vivo imaging with xfOCM of amyloid plaque in a mouse model of Alzheimer’s disease (AD), without administration of extrinsic contrast agents. By implementing a 3-D image segmentation algorithm, we further show how xfOCM can be employed to perform a label-free ex vivo transversal study of the development of amyloid plaque in the mouse brain. The high acquisition speed obtained from multiplexing depth acquisition is further exploited to image cerebral blood flow. We first analyze the Doppler frequency spectrum measured by OCM when scatterers such as erythrocytes flow through the system’s focus and show how it relates to the axial and lateral flow velocity components. This then enables quantitative blood flow imaging with xfOCM, with in an unprecedented combination of 3-D resolution and acquisition speed. We apply this technique to label-free in vivo angiography and quantitative blood flow imaging in the murine brain. xfOCM achieves deep tissue penetration due to the use of a near-infrared spectrum. The study of cell cultures, however, does not require such deep penetration, but instead could benefit from higher resolution. For these applications, we introduce visible spectrum OCM (visOCM), enabling label-free and long-term imaging of living cell cultures with 3-D sub-micron resolution. visOCM allows cell morphology to be imaged in thin single layer cultures, but also in thicker three-dimensional cultures and organotypic slices, for which no label-free tomographic microscopy exists to date.

EPFL2013

Interferometric synthetic aperture microscopy for extended focus optical coherence microscopy

Arno Pino Bouwens, Séverine Coquoz, Jérôme Extermann, Theo Lasser, Paul James Marchand

Optical coherence microscopy (OCM) is an interferometric technique providing 3D images of biological samples with micrometric resolution and penetration depth of several hundreds of micrometers. OCM differs from optical coherence tomography (OCT) in that it uses a high numerical aperture (NA) objective to achieve high lateral resolution. However, the high NA also reduces the depth-of-field (DOF), scaling with 1/NA2. Interferometric synthetic aperture microscopy (ISAM) is a computed imaging technique providing a solution to this trade-off between resolution and DOF. An alternative hardware method to achieve an extended DOF is to use a non-Gaussian illumination. Extended focus OCM (xfOCM) uses a Bessel beam to obtain a narrow and extended illumination volume. xfOCM detects back-scattered light using a Gaussian mode in order to maintain good sensitivity. However, the Gaussian detection mode limits the DOF. In this work, we present extended ISAM (xISAM), a method combining the benefits of both ISAM and xfOCM. xISAM uses the 3D coherent transfer function (CTF) to generalize the ISAM algorithm to different system configurations. We demonstrate xISAM both on simulated and experimental data, showing that xISAM attains a combination of high transverse resolution and extended DOF which has so far been unobtainable through conventional ISAM or xfOCM individually.

2017

Structural and functional brain imaging using extended-focus optical coherence tomography and microscopy

Paul James Marchand

Neuroimaging techniques aim at revealing the anatomy and functional organisation of cerebral structures. Over the past decades, functional magnetic resonance imaging (fMRI) has revolutionized our understanding of human cerebral physiology through its ability to probe neural activity throughout the entire brain in a non-invasive fashion. Nevertheless, despite recent technological improvements, the spatial resolution of fMRI remains limited to a few hundreds of microns, restricting its use to macroscopic studies. Microscopic imaging solutions have been proposed to circumvent this limitation and have enabled revealing the existence of various cerebral structures, such as neuronal and vascular networks and their contribution to information processing and blood flow regulation within the brain. Optical imaging has proven, through its increased resolution and available contrast mechanisms, to be a valuable complement to fMRI for cellular-scale imaging. In this context, we demonstrate here the capabilities of an extension of optical coherence tomography, termed extended-focus optical coherence tomography (xf-OCT), in imaging cerebral structure and function at high resolution and very high acquisitions rates. Optical coherence tomography is an interferometric imaging technique using a low-coherence illumination source to provide fast, three-dimensional imaging of the back-scattering of tissue and cells. By multiplexing the interferometric ranging over several spectral channels, Fourier-domain OCT performs depth-resolved imaging at very high acquisition rates and high sensitivity. Increasing the lateral resolution of optical systems typically reduces the available depth-of-field and thus hampers this depth multiplexing advantage of OCT. Extended-focus systems aim at alleviating this trade-off between imaging depth and lateral resolution through the use of diffraction-less beams such as Bessel beams, providing high resolution imaging over large depths. The xf-OCT system therefore combines fast acquisition rates and high resolution, both characteristics required to image and study the structure and function of microscopic constituents of cerebral tissue. In this work, we performed functional brain imaging using the ability of xf-OCT to obtain quantita- tive measurements of blood flow in the brain. Changes in blood velocity evoked by neuronal activation were monitored and maps of hemodynamic activity were generated by adapting tools routinely used in fMRI to xf-OCT imaging. Additionally, three novel xf-OCT instruments are presented, wherein the advantages of different spectral ranges are exploited to reach specific imaging parameters. The increased contrast and resolution afforded by an illumination in the visible spectral range was used in two extended-focus optical coherence microscopy (xf-OCM) implementations for subcellular imaging of ex-vivo brain slices and cellular imaging of neurons, capillaries and myelinated axons in the superficial cortex in-vivo. Subsequently, an xf-OCT system is presented, operating in the infrared spectral range, wherein the reduced scattering enabled imaging the smallest capillaries deep in the murine cortex in-vivo.