High-resolution 3-D imaging of living cells in suspension using confocal axial tomography

ArticleinBiotechnology Journal 3(1):53-62 · January 2008with6 Reads
DOI: 10.1002/biot.200700188 · Source: PubMed
Conventional flow cytometry (FC) methods report optical signals integrated from individual cells at throughput rates as high as thousands of cells per second. This is further combined with the powerful utility to subsequently sort and/or recover the cells of interest. However, these methods cannot extract spatial information. This limitation has prompted efforts by some commercial manufacturers to produce state-of-the-art commercial flow cytometry systems allowing fluorescence images to be recorded by an imaging detector. Nonetheless, there remains an immediate and growing need for technologies facilitating spatial analysis of fluorescent signals from cells maintained in flow suspension. Here, we report a novel methodological approach to this problem that combines micro-fluidic flow, and microelectrode dielectric-field control to manipulate, immobilize and image individual cells in suspension. The method also offers unique possibilities for imaging studies on cells in suspension. In particular, we report the system's immediate utility for confocal "axial tomography" using micro-rotation imaging and show that it greatly enhances 3-D optical resolution compared with conventional light reconstruction (deconvolution) image data treatment. That the method we present here is relatively rapid and lends itself to full automation suggests its eventual utility for 3-D imaging cytometry.
    • "For example, Vishnyakov et al. [14] produced images of a lymphocyte. Renaud et al. [11] produced images of SW13 cells. The approaches used apply, or could be adapted to apply, to the attenuation and refractive index of the volume illuminated by the light. "
    [Show abstract] [Hide abstract] ABSTRACT: We introduce and experimentally validate a computational imaging technique that employs confocal scanning and coherent detection in the Fourier domain. We show how this method may be used to to-mographically reconstruct attenuation, aberration, and even occlusion. We also show how these image parameters may be combined with the conventional confocal image reconstruction of the object reflectivity. We demonstrate the method experimentally by imaging a sample consisting of an occlusion above a mirror of varying reflectivity.
    Article · May 2010
    • "The fact that biology is a 3D problem was once more underlined by Spencer Shorte (Pasteur Institute, Paris, France), who presented two techniques allowing imaging of cells in flow suspension: one commercial system combining flow cytometry with two-dimensional (2D) fluorescence imaging (George et al., 2004; McGrath et al., 2008 ); and the second combing microfluidic flow, dielectric field cell trapping and 3D imaging. The dielectric trap is used to capture cells inside a flow chamber where they can be imaged in high resolution by a method called 'microrotation imaging' and reconstructed by 3D confocal axial tomography (Renaud et al., 2008). The unique power of conventional flow cytometry is high-throughput single-cell sampling at rates of thousands of cells per minute, and even per second. "
    Article · May 2009
    • "The piezo step in through-stack axial imaging was 100 nm and xy resolution was 127 nm. More details about z-stack acquisition can be found in (Renaud et al., 2008). The choice of the appropriated PSF is crucial. "
    [Show abstract] [Hide abstract] ABSTRACT: Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.
    Full-text · Article · Apr 2009
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