Phase-Retrieved Tomography enables imaging of a Tumor Spheroid in Mesoscopy Regime

Optical tomographic imaging of biological specimen bases its reliability on the combination of both accurate experimental measures and advanced computational techniques. In general, due to high scattering and absorption in most of the tissues, multi view geometries are required to reduce diffuse halo and blurring in the reconstructions. Scanning processes are used to acquire the data but they inevitably introduces perturbation, negating the assumption of aligned measures. Here we propose an innovative, registration free, imaging protocol implemented to image a human tumor spheroid at mesoscopic regime. The technique relies on the calculation of autocorrelation sinogram and object autocorrelation, finalizing the tomographic reconstruction via a three dimensional Gerchberg Saxton algorithm that retrieves the missing phase information. Our method is conceptually simple and focuses on single image acquisition, regardless of the specimen position in the camera plane. We demonstrate increased deep resolution abilities, not achievable with the current approaches, rendering the data alignment process obsolete.Comment: 21 pages, 5 figure

3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms

Assembling models of embryo development: Image analysis and the construction of digital atlases

Digital atlases of animal development provide a quantitative description of morphogenesis, opening the path toward processes modeling. Prototypic atlases offer a data integration framework where to gather information from cohorts of individuals with phenotypic variability. Relevant information for further theoretical reconstruction includes measurements in time and space for cell behaviors and gene expression. The latter as well as data integration in a prototypic model, rely on image processing strategies. Developing the tools to integrate and analyze biological multidimensional data are highly relevant for assessing chemical toxicity or performing drugs preclinical testing. This article surveys some of the most prominent efforts to assemble these prototypes, categorizes them according to salient criteria and discusses the key questions in the field and the future challenges toward the reconstruction of multiscale dynamics in model organisms

Reconstruction of neuronal activity and connectivity patterns in the zebrafish olfactory bulb

In the olfactory bulb (OB), odors evoke distributed patterns of activity across glomeruli that are reorganized by networks of interneurons (INs). This reorganization results in multiple computations including a decorrelation of activity patterns across the output neurons, the mitral cells (MCs). To understand the mechanistic basis of these computations it is essential to analyze the relationship between function and structure of the underlying circuit. I combined in vivo twophoton calcium imaging with dense circuit reconstruction from complete serial block-face electron microscopy (SBEM) stacks of the larval zebrafish OB (4.5 dpf) with a voxel size of 9x9x25nm. To address bottlenecks in the workflow of SBEM, I developed a novel embedding and staining procedure that effectively reduces surface charging in SBEM and enables to acquire SBEM stacks with at least a ten-fold increase in both, signal-to-noise as well as acquisition speed. I set up a high throughput neuron reconstruction pipeline with >30 professional tracers that is available for the scientific community (ariadne-service.com). To assure efficient and accurate circuit reconstruction, I developed PyKNOSSOS, a Python software for skeleton tracing and synapse annotation, and CORE, a skeleton consolidation procedure that combines redundant reconstruction with targeted expert input. Using these procedures I reconstructed all neurons (>1000) in the larval OB. Unlike in the adult OB, INs were rare and appeared to represent specific subtypes, indicating that different sub-circuits develop sequentially. MCs were uniglomerular whereas inter-glomerular projections of INs were complex and biased towards groups of glomeruli that receive input from common types of sensory neurons. Hence, the IN network in the OB exhibits a topological organization that is governed by glomerular identity. Calcium imaging revealed that the larval OB circuitry already decorrelates activity patterns evoked by similar odors. The comparison of inter-glomerular connectivity to the functional interactions between glomeruli indicates that pattern decorrelation depends on specific, non-random inter-glomerular IN projections. Hence, the topology of IN networks in the OB appears to be an important determinant of circuit function

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Light-sheet microscopy: a tutorial

This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.Peer ReviewedPostprint (published version

Development of novel imaging tools for selected biomedical applications

In the quest for better and faster images of cellular and subcellular structures, biology-oriented optical microscopes have advanced significantly in the last few decades. Novel microscopy techniques such as non-linear microscopy (NLM), including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy, and light-sheet fluorescence microscopy (LSFM) are emerging as alternatives that overcome some the intrinsic limitations of standard microscopy systems. In this thesis I aimed to advance such techniques even more, and combine them with other photonic technologies to provide novel tools that would help to address complex biological questions. This thesis is organized in two main parts. The first part is dedicated to applications involving femtosecond lasers that are employed for precise microsurgery. For that, damage assessment methodologies based on NLM were developed and tested in relevant biomedical models. In the second part, wavefront engineering methods were employed to enhance the imaging capabilities of light-sheet microscopy systems. These novel methodologies were tested as well in relevant biological applications. This thesis is, therefore, organized as follows: In chapter 1, a brief and comprehensive review of the basic microscopy techniques employed in this thesis is presented, together with the challenges and achievements of this thesis in sequential order. In chapter 2, a multimodal imaging methodology for the assessment of laser induced collateral damage is presented. This was specifically developed for the control of the damage in femtosecond-laser dissection of single axons within a living Caenorhabditis elegans (C. elegans). Here, it is shown that collateral damages at the level of the myosin structure of the muscles adjacent to the axon, can be readily detected. In chapter 3, the optimized multimodal methodology developed in the chapter 2 was employed for minimally invasive dissection of axons of D-type motoneurons in C elegans. Here, a microfluidic chip for C elegans immobilization and a detailed protocol was employed to evaluate the axon regeneration of such neurons. The potential of such platform for testing drugs with regeneration-enhancing capabilities is also presented. In chapter 4, a novel use of TPEF microscopy is presented to characterize and fine tune the laser for photodisruption of excised human crystalline lens samples. In chapter 5, a thorough description of the implementation of a multimodal Digital Scanned Light-Sheet Microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes, is presented. The enhanced capabilities of the developed system is evaluated using in vivo C. elegans samples and multicellular tumor spheroids In chapter 6, the development of a completely new concept in light sheet-based imaging is presented. This is based on the extension of the depth-of-field of the lens in the emission path of the microscope by using wavefront coding (WFC) techniques. Furthermore, I demonstrate the application of the developed methodology for fast volumetric imaging of living biological specimens and 3D particle tracking

Contrast Mechanisms & Wavefront Control in Coherent Nonlinear Microscopy

This Thesis deals with theoretical and experimental aspects of coherent nonlinear microscopy, with a special attention given to contrast mechanisms in Third Harmonic Generation (THG) microscopy.Ce travail de thèse présente une étude théorique et expérimentale de la microscopie non-linéaire cohérente pour l’étude de systèmes biologiques.Dans un premier temps, nous avons étudié l’origine des signaux et les mécanismes de contrastes en microscopie non-linéaire cohérente. En particulier, nous avons analysé l’influence de la microstructure de l’échantillon sur le signal de troisième harmonique rayonné. Nous illustrons cette discussion par une étude sur les contrastes endogènes de la cornée humaine. Dans un second temps, nous nous sommes intéressés à diverses méthodes de contrôle de front d’onde afin de moduler le contraste des images. Nous avons effectué par une étude théorique du signal obtenus à l’aide de modes d’excitation spatiale non-Gaussiens, puis avons implémenté deux systèmes de modulation du front d’onde : un modulateur acousto-optique permettant d’obtenir une profondeur de champ étendue en microscopie par fluorescence excitée à deux photons, et un miroir déformable permettant d’effectuer une correction dynamique des aberrations en microscope par génération de troisième harmonique. Enfin, nous nous sommes intéressés à l’application de la microscopie non-linéaire cohérente pour l’observation du développement précoce d’organismes modèles en embryologie: la drosophile et le poisson zèbre

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model