In vivo label-free three-dimensional imaging of zebrafish vasculature with optical projection tomography

Politecnico di Milano, Dipartimento di Fisica, Piazza Leonardo da Vinci 32, Milan, 20133, Italy.
Journal of Biomedical Optics (Impact Factor: 2.86). 10/2011; 16(10):100502. DOI: 10.1117/1.3640808
Source: PubMed


We introduce flow optical projection tomography, an imaging technique capable of visualizing the vasculature of living specimens in 3-D. The method detects the movement of cells in the bloodstream and creates flow maps using a motion-analysis procedure. Then, flow maps obtained from projection taken at several angles are used to reconstruct sections of the circulatory system of the specimen. We therefore demonstrate an in vivo, 3-D optical imaging technique that, without the use of any labeling, is able to reconstruct and visualize the vascular network of transparent and weakly scattering living specimens.

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    • "The zebrafish is anesthetized with Tricaine mesylate, mounted in a 2-mm-diam fluorinated ethylene propylene (FEP) tube (FT2X3, Adtech Polymer Engineering, UK) with 1.5% low melting point agarose and then immersed in a water cell. [19] "
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    ABSTRACT: The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.
    Proceedings of SPIE - The International Society for Optical Engineering 02/2013; 8580. DOI:10.1117/12.2002082 · 0.20 Impact Factor
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    • "For instance, Ertürk et al. proposed a new tissue clearing procedure that allows achieving cellular resolution imaging of axon up to 4 mm deep in an unsectioned spinal cord of adult GFP-M mice [11]. Moreover, millimeter scale mesoscopic imaging techniques such as selective plane illumination microscopy [12] and optical projection tomography [13] have been developed recently and applied successfully to image millimeter thick cleared samples. These techniques provide unique tools for applications, such as developmental biology. "
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    ABSTRACT: We investigated the potential of fluorescence molecular tomography to image ex vivo samples collected from a large animal model, in this case, a dog spine. Wide-field time-gated fluorescence tomography was employed to assess the impact of multiview acquisition, data type, and intrinsic optical properties on the localization and quantification accuracy in imaging a fluorescent inclusion in the intervertebral disk. As expected, the TG data sets, when combining early and late gates, provide significantly better performances than the CW data sets in terms of localization and quantification. Moreover, the use of multiview imaging protocols led to more accurate localization. Additionally, the incorporation of the heterogeneous nature of the tissue in the model to compute the Jacobians led to improved imaging performances. This preliminary imaging study provides a proof of concept of the feasibility of quantitatively imaging complex ex vivo samples nondestructively and with short acquisition times. This work is the first step towards employing optical molecular imaging of the spine to detect and characterize disc degeneration based on targeted fluorescent probes.
    International Journal of Biomedical Imaging 11/2012; 2012:942326. DOI:10.1155/2012/942326
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    • "Confocal or two-photon microscopy has been extensively employed for imaging zebrafish a few days post-fertilization (dpf) (Isogai et al 2001, Megason and Fraser 2003, Das et al 2003), requiring injection of a fluorescent probe or the use of fluorescent reporters. Other label-free optical imaging techniques, such as optical coherence tomography (Kagemann et al 2008), selective plane illumination microscopy (Arrenberg et al 2010) or optical projection tomography (Bassi et al 2011), have also been recently used for imaging zebrafish at early stages. Although one zebrafish mutant strain has been recently established, which stays transparent throughout its life (White et al 2008), other commonly used zebrafish strains lose their transparency starting at approximately 14 dpf and are thus not accessible for high-resolution optical imaging approaches at adult stages. "
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    ABSTRACT: Zebrafish has emerged as an excellent vertebrate model organism for studies of evolution, development and disease. Due to its external development and optical transparency in embryonic stages, zebrafish offers a major advantage over other vertebrate model organisms by being amenable for microscopic studies of biological processes within their natural environment directly in the living organism. However, commonly used zebrafish strains lose their transparency within their first two weeks of development and thus are no longer accessible for optical imaging approaches at juvenile or adult stages. In this study we successfully apply optoacoustic imaging for non-invasive three-dimensional imaging of adult zebrafish. Since optoacoustics does not necessarily require labeling, but can instead rely on the intrinsic tissue contrast, this imaging method has the potential to become a versatile tool for developmental studies from juvenile to adult stages in the intact zebrafish.
    Physics in Medicine and Biology 10/2012; 57(22):7227-7237. DOI:10.1088/0031-9155/57/22/7227 · 2.76 Impact Factor
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