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.
- SourceAvailable from: Alain Trouvé
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- "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). "
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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- "cells flow freely in suspension apart from the force included by the electric field of the cell rotator. However, careful image acquisition improves the rotation stability 5 : an example of a situation where the rotation stability is satisfactory is provided by Renaud et al (2008) who demonstrated that the cell rotator is capable of reproducing high correlation images of the cell after a complete rotation of 360 • (with 90 • stepwise). With sufficient rotation stability, the acquired images can be accurately aligned using, for instance, a 2D cross-correlation method with a prior constraint (Palander 2007). "
ABSTRACT: Micro-rotation confocal microscopy is a novel optical imaging technique which employs dielectric fields to trap and rotate individual cells to facilitate 3D fluorescence imaging using a confocal microscope. In contrast to computed tomography (CT) where an image can be modelled as parallel projection of an object, the ideal confocal image is recorded as a central slice of the object corresponding to the focal plane. In CT, the projection images and the 3D object are related by the Fourier slice theorem which states that the Fourier transform of a CT image is equal to the central slice of the Fourier transform of the 3D object. In the micro-rotation application, we have a dual form of this setting, i.e. the Fourier transform of the confocal image equals the parallel projection of the Fourier transform of the 3D object. Based on the observed duality, we present here the dual of the classical filtered back projection (FBP) algorithm and apply it in micro-rotation confocal imaging. Our experiments on real data demonstrate that the proposed method is a fast and reliable algorithm for the micro-rotation application, as FBP is for CT application.Inverse Problems 11/2008; 25(1):015006. DOI:10.1088/0266-5611/25/1/015006 · 1.80 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