High-resolution 3-D imaging of living cells in suspension using confocal axial tomography
ABSTRACT 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.
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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.Journal of Microscopy 04/2009; 233(3):404-16. DOI:10.1111/j.1365-2818.2009.03128.x · 2.15 Impact Factor
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ABSTRACT: Flow cytometry provides a high throughput, multi-dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z-resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.Journal of Biophotonics 03/2015; 9999(9999). DOI:10.1002/jbio.201300196 · 3.86 Impact Factor
Conference Paper: Micro-rotation imaging deconvolution[Show abstract] [Hide abstract]
ABSTRACT: Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of slices of non adherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the 3D position of every slice with respect to the frame, the reconstruction of the cell from the positioned slices remains a problem that this paper address. In our context, 3D spatially-varying PSF and missing data are the two main particularities of this problem. Experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume and also to deal with missing data.Biomedical Imaging: From Nano to Macro, 2008. ISBI 2008. 5th IEEE International Symposium on; 06/2008