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: In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rotate a cell both in the imaging plane and also in a perpendicular plane allowing tomographic imaging of the trapped living cell. The integrated device allows easy operation and by combining it with high-resolution confocal microscopy we show for the first time that the pattern of rotation can give information regarding the sub-cellular composition of a rotated cell.Lab on a Chip 02/2014; · 5.70 Impact Factor
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ABSTRACT: In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ( and sperm) or frozen-thawed semen ( sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ( sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.Asian Australasian Journal of Animal Sciences 01/2010; 23(1). · 0.64 Impact Factor