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

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 {\mu}m axial and 0.4 {\mu}m lateral resolution maintained over a depth of 40 {\mu}m, while preserving the advantages of Fourier domain OCM. Our method uses an ultra-broad spectrum from a super- continuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the method 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 mouse brain tissue.Comment: 15 pages, 7 figure

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

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

Discours sur la fin

Approche du genre littéraire apocalyptique au travers d'une étude comparative entre IV Esdras et II Baruch

High-speed phase-shifting common-path quantitative phase imaging with a piezoelectric actuator

We present a phase-shifting quantitative phase imaging technique providing high temporal and spatial phase stability and high acquisition speed. A piezoelectric microfabricated phase modulator allows tunable modulation frequencies up to the kHz range. After assessing the quantitative phase accuracy with technical samples, we demonstrate the high acquisition rate while monitoring cellular processes at temporal scales ranging from milliseconds to hours

Interferometric synthetic aperture microscopy for extended focus optical coherence microscopy

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

Large-scale analysis of high-speed atomic force microscopy data sets using adaptive image processing

Modern high-speed atomic force microscopes generate significant quantities of data in a short amount of time. Each image in the sequence has to be processed quickly and accurately in order to obtain a true representation of the sample and its changes over time. This paper presents an automated, adaptive algorithm for the required processing of AFM images. The algorithm adaptively corrects for both common one-dimensional distortions as well as the most common two-dimensional distortions. This method uses an iterative thresholded processing algorithm for rapid and accurate separation of background and surface topography. This separation prevents artificial bias from topographic features and ensures the best possible coherence between the different images in a sequence. This method is equally applicable to all channels of AFM data, and can process images in seconds

Schematic of the visOCM setup for <i>C. elegans</i> imaging.

<p>Light from a laser source with a broad spectrum in the visible range (<b>A</b>, inset) is collimated by lens L1 and split by beam-splitter BS1 into a sample (green) and reference (blue) arm. In the sample arm, the axicon lens generates a Bessel-like illumination beam which is then guided to the tube lens (TL) and objective by the X-Y galvo-scanner unit. The back-reflected light (red) from the sample (<b>B</b>, inset) is recombined with the reference arm by beam-splitter BS2 and focused by L2 into the detection fiber. Finally, the spectrometer (<b>C</b>, inset), records the interference pattern which is processed to yield a depth profile of the <i>C. elegans</i> structure. The data processing steps are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181676#pone.0181676.s001" target="_blank">S1 Fig</a>. Scale bars: 25 μm.</p

Anatomy of the <i>C. elegans</i> as revealed by visOCM.

<p><b>(A, B)</b><i>En face</i> projections at two different depths, and <b>(C)</b> side view at the location highlighted in <b>(B)</b>. Scale bars indicate 50 μm. <b>(D)</b> Top: A 3D rendered model of the head with the pharynx highlighted in green. Bottom: Maximum-intensity projection through the entire animal’s head. <b>(E)</b> <i>En face</i> view (top) and corresponding transverse sections (bottom), with the lumen of the intestine highlighted in yellow. <b>(F)</b> Zoom regions of the reproductive system showing germ cells, oocytes, spermatheca, embryos and the vulva. The 3D sub-micrometer resolution and the intrinsic contrast of our technique enable a clear and detailed visualization of tissue structures down to the sub-cellular level (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181676#pone.0181676.s003" target="_blank">S2 Video</a>).</p