Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos.

The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.

ANISOTROPIC PLATE DIFFUSION FILTERING FOR DETECTION OF CELL MEMBRANES IN 3D MICROSCOPY IMAGES.

We propose an anisotropic diffusion method to denoise and aid the reconstruction of planar objects in three-dimensional images. The contribution of this paper is the development of a planarity function characterizing plate-like structures using an image Hessian's eigensystem. We then construct a diffusion tensor for anisotropically smoothing plates and satisfying necessary scale-space properties. Our method finds applications in improving the fidelity of highly noisy cell membrane images from confocal microscopy. In dense cellular regions, cell membranes assume linear shapes (planar) between neighbors. The imaging process makes cell membranes appear as diffuse structures owing to the non-uniform fluorescent marker distribution, point-spread function of the optics, and anisotropic voxel resolution which make automatic cell segmentation difficult. We apply diffusion filtering to identify and enhance membranes. We demonstrate the use of our methods on 3D cell membrane images of a zebrafish embryo acquired using fluorescent microscopy and quantify the improvement in image quality.

VARIATIONAL LEVEL-SET WITH GAUSSIAN SHAPE MODEL FOR CELL SEGMENTATION.

In analysis of microscopy based images, a major challenge lies in splitting apart cells that appear to overlap because they are too densely packed. This task is complicated by the physics of the image acquisition that causes large variations in pixel intensities. Each image typically contains thousands of cells with each cell having a different orientation, size and intensity histogram. In this paper, a spatial intensity model of a nucleus is incorporated into [1] to aid cell segmentation from microscopy datasets. An energy functional is defined and with it the spatial intensity distribution of a nuclei is modeled as a Gaussian distribution with constant intensity background. Experimental results on a variety of microscopic data validate its effectiveness.