Four Dimensional Image Registration For Intravital Microscopy

Increasingly the behavior of living systems is being evaluated using intravital microscopy since it provides subcellular resolution of biological processes in an intact living organism. Intravital microscopy images are frequently confounded by motion resulting from animal respiration and heartbeat. In this paper we describe an image registration method capable of correcting motion artifacts in three dimensional fluorescence microscopy images collected over time. Our method uses 3D B-Spline non-rigid registration using a coarse-to-fine strategy to register stacks of images collected at different time intervals and 4D rigid registration to register 3D volumes over time. The results show that our proposed method has the ability of correcting global motion artifacts of sample tissues in four dimensional space, thereby revealing the motility of individual cells in the tissue

Vascular patterning of subcutaneous mouse fibrosarcomas expressing individual VEGF isoforms can be differentiated using angiographic optical coherence tomography

Subcutaneously implanted experimental tumors in mice are commonly used in cancer research. Despite their superficial location, they remain a challenge to image noninvasively at sufficient spatial resolution for microvascular studies. Here we evaluate the capabilities of optical coherence tomography (OCT) angiography for imaging such tumors directly through the murine skin in-vivo. Datasets were collected from mouse tumors derived from fibrosarcoma cells genetically engineered to express only single splice variant isoforms of vascular endothelial growth factor A (VEGF); either VEGF120 or VEGF188 (fs120 and fs188 tumors respectively). Measured vessel diameter was found to be significantly (p<0.001) higher for fs120 tumors (60.7±4.9μm) compared to fs188 tumors (45.0±4.0μm). The fs120 tumors also displayed significantly higher vessel tortuosity, fractal dimension and density. The ability to differentiate between tumor types with OCT suggests that the visible abnormal vasculature is representative of the tumor microcirculation, providing a robust, non-invasive method for observing the longitudinal dynamics of the subcutaneous tumor microcirculation

Modeling Oxygen Transport in Three-Dimensional Capillary Networks

The purpose of this thesis was to examine how the use of real 3-dimensional (3D) capillary network geometries affect models of oxygen transport to tissue. Software was developed to reconstruct microvascular geometry in 3D from intravital video. Characterization of 3D reconstructions demonstrated that capillary density, length and capillary diameter were consistent with previous findings. Using reconstructed capillary networks a strategy was devised that utilized red blood cell (RBC) supply rate (SR) as a metric for flow modeling. Applying the RBC SR based flow model on baseline and perturbed flow conditions demonstrated that RBC SR is a major determinant of oxygen delivery that is insensitive to changes in flow distribution. The resulting flow solutions were used for comparing oxygen transport in 3D networks and synthetic parallel arrays. A variety of physiological conditions were simulated and it was determined that parallel arrays resulted in oxygen transport solutions with higher mean PO2 due to homogeneous distribution of vessels in the volume. Lastly, to investigate oxygen transport in a complex pathology a model of sepsis was used to investigate how incremental perfusion loss, consumption increase and change in RBC SR affect oxygen delivery. It was shown that perfusion loss did not markedly impair oxygen delivery provided that RBC SR was maintained. These results have improved our understanding of oxygen transport to tissue in normal and diseased conditions; the use of reconstructed networks and measurements of blood flow & oxygen saturation in computer models provides different solutions than those using statistical averages and synthetic networks

Optically gated beating-heart imaging

The constant motion of the beating heart presents an obstacle to clear optical imaging, especially 3D imaging, in small animals where direct optical imaging would otherwise be possible. Gating techniques exploit the periodic motion of the heart to computationally "freeze" this movement and overcome motion artefacts. Optically gated imaging represents a recent development of this, where image analysis is used to synchronize acquisition with the heartbeat in a completely non-invasive manner. This article will explain the concept of optical gating, discuss a range of different implementation strategies and their strengths and weaknesses. Finally we will illustrate the usefulness of the technique by discussing applications where optical gating has facilitated novel biological findings by allowing 3D in vivo imaging of cardiac myocytes in their natural environment of the beating heart

Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

International audience: Dynamics of the lung immune system at a microscopic level are largely unknown because of inefficient methods to rid chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process

Three-dimensional imaging and reconstruction of the whole ovary and testis: a new frontier for the reproductive scientist.

AbstractThe 3D functional reconstruction of a whole organ or organism down to the single cell level and to the subcellular components and molecules is a major future scientific challenge. The recent convergence of advanced imaging techniques with an impressively increased computing power allowed early attempts to translate and combine 2D images and functional data to obtain in-silico organ 3D models. This review first describes the experimental pipeline required for organ 3D reconstruction: from the collection of 2D serial images obtained with light, confocal, light-sheet microscopy or tomography, followed by their registration, segmentation and subsequent 3D rendering. Then, we summarise the results of investigations performed so far by applying these 3D image analyses to the study of the female and male mammalian gonads. These studies highlight the importance of working towards a 3D in-silico model of the ovary and testis as a tool to gain insights into their biology during the phases of differentiation or adulthood, in normal or pathological conditions. Furthermore, the use of 3D imaging approaches opens to key technical improvements, ranging from image acquisition to optimisation and development of new processing tools, and unfolds novel possibilities for multidisciplinary research