Microscopy and image acquisition All specimens were mounted in Vectashield. To preserve the three dimensional nuclear structure as much as possible, a thin spacer was drawn with a Dako pen around the embryos before covering them with a 170 um thick coverslip. Imaging was performed with an inverted Zeiss LSM 510 confocal microscope with a 63X oil immersion objective. http://www.selleckchem.com/products/Dasatinib.html The z stacks were acquired using a frame size of 512 512, a pixel depth of 8 bits, and 0. 371 um z steps, with sequential multitrack scanning using the 488, 543, and 633 nm wavelengths of the lasers. Computational image analysis All embryos were first visually analyzed with the LSM510 software, step by Inhibitors,Modulators,Libraries step through the confocal z stacks, and with the help of 3D reconstructions using AMIRA software.

Except for the 1 cell stage embryos, which presented a peculiar nuclear organization, we then analyzed all the preimplantation embryos with Inhibitors,Modulators,Libraries the semi automated image processing and analysis tools described hereafter. These tools are based on the ITK library interfaced with Python scripting language. In each case, the LSM image files were first imported with the Bio Formats library hen the color channels were split into separate 3D data sets and upsampled to get a threefold increase in the number of pixels along the z axis with an isotropic voxel size. Images were then processed to get segmented, labeled objects. To check the efficiency of the segmentation pro cedures, segmented images were superimposed on their original grayscale image using either macros developed with the ImageJ software or the 3D object analysis from Fiji software.

Segmentation of nuclei Segmentation of the nuclei in the DNA channel was a critical step because it defined the regions of interest where we looked for centromeric and pericentro meric structures in our 3D data sets. Since most voxels corresponded to the background of the images, a 3D binary mask was first determined by a threshold Inhibitors,Modulators,Libraries method largely Inhibitors,Modulators,Libraries used in astronomy it analyzes only the back ground intensities and assigns the intensity value as the lower threshold. To get the best mask, the weighting factor f was used as the signal to noise ratio in the embryos. This SNR value was generally between 1 and 2 under our image acquisition conditions. when it was outside this range, we used the closest limit of this interval.

Next, nuclei were extracted from the binary mask with an a priori method based on their size and shape a combination of 2D and 3D attribute Inhibitors,Modulators,Libraries opening transfor mations was applied to remove the smallest objects. Connected voxels representing nuclei were then identi fied with label object representation and manipulation filters . 3D morphological opening and closing merely trans formations were applied to fill and smooth the rough la beled objects.