Article

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

Institut Pasteur, Plate-forme d'Imagerie Dynamique, Imagopole, Paris, France.
Biotechnology Journal (Impact Factor: 3.71). 01/2008; 3(1):53-62. DOI: 10.1002/biot.200700188
Source: PubMed

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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    • "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). "
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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). "
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