Segmentation and Quantitative Analysis of Individual Cells in Developmental Tissues

Abstract

Image analysis is vital for extracting quantitative information from biological images and is used extensively, including investigations in developmental biology. The technique commences with the segmentation (delineation) of objects of interest from 2D images or 3D image stacks and is usually followed by the measurement and classification of the segmented objects. This chapter focuses on the segmentation task and here we explain the use of ImageJ, MIPAV (Medical Image Processing, Analysis, and Visualization), and VisSeg, three freely available software packages for this purpose. ImageJ and MIPAV are extremely versatile and can be used in diverse applications. VisSeg is a specialized tool for performing highly accurate and reliable 2D and 3D segmentation of objects such as cells and cell nuclei in images and stacks.

Appendix

Sample preparation and image acquisition. Two biological samples were used in this study. One was an early somite stage (E8.0–8.5) mouse embryo obtained from an NIH Swiss female mouse. The embryo was fixed with 2 % paraformaldehyde/phosphate buffered saline (PBS) for 30 min at room temperature and stained with Oregon Green 488 Phalloidin (O-7466, Molecular Probes, Eugene, OR, USA) after treatment with 0.1 M glycine/0.2 % Triton X-100/PBS for 10 min. Phalloidin predominantly labels filamentous actin found abundantly at the cell surface underlying the cell membrane. The embryo was counter stained with the DNA dye 4′,6-diamidino-2–phenylindole (DAPI). The posterior primitive streak region of the embryos was manually dissected and mounted with SlowFade Light Antifade kit (S-7461, Molecular Probes) on a glass slide. Images were acquired using a 40×, 1.3 numerical aperture oil objective lens, and pinhole of one airy unit on an LSM 510 confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA). Excitation was with a 488-nm laser light and emitted light between 500 and 550 nm was acquired. Thus, the effective thickness of the optical sections was 425 nm at the coverslip but increased further away from the coverslip. Pixel size was 0.14 μm in the x and y dimensions. DAPI was excited with two photon excitation at 780 nm and emitted light between 420 and 480 nm was collected.

The other biological sample used in this chapter was a 3D cell culture model of early breast tumorigenesis. MCF-10A.B2 a human mammary epithelial cell line was grown on basement membrane extract (Trevigen Inc., Gaithersburg, MD) for 20 days. MCF-10A.B2 cells express a synthetic ligand-inducible active ErbB2 variant. To induce tumorigenesis the synthetic ligand (AP1510; ARIAD Pharmaceuticals Inc)) was added for the final 10 days of culture. The acini structures formed by growth in 3D culture were fixed in 2 % paraformaldehyde/PBS for 20 min at room temperature and permeabilized by a 10 min incubation in 0.5 % Triton X-100 at 4 °C. Following three 15 min rinses in 100 mM glycine/PBS, the acini were blocked in IF buffer (0.1 % bovine serum albumin/0.2 % Triton X-100/0.05 % Tween-20/PBS) containing 10 % fetal bovine serum (Invitrogen) for 1 h. The cells were then incubated overnight in anti-integrin alpha 6 antibody (Chemicon International). Integrin alpha 6 stains for basolateral polarity, it stains the cell surface membrane with strong basal and weaker lateral staining. After three 20 min washes in IF buffer the cells were incubated in Alexa Fluor 488 donkey anti-rat (Invitrogen). Samples were mounted in DAPI containing VECTASHIELD mounting media (Vector Laboratories Ltd.) after three further 20 min washes in IF buffer. Images were acquired using a 63× 1.4-NA oil objective lens on an LSM 510 confocal microscope.