Confocal scanning laser microscopy with complementary 3D image analysis allows quantitative studies of functional state of ionoregulatory cells in the nile tilapia (Oreochromis niloticus) following salinity challenge

Abstract

The development of a novel three-dimensional image analysis technique of stacks generated by confocal laser scanning microscopy is described allowing visualization of mitochondria-rich cells (MRCs) in the seawater-adapted Nile tilapia in relation to their spatial location. This method permits the assessment and classification of both active and nonactive MRCs based on the distance of the top of the immunopositive cell from the epithelial surface. In addition, this technique offers the potential for informative and quantitative studies, for example, densitometric and morphometric measurements based on MRC functional state. Confocal scanning laser microscopy used with triple staining whole-mount immunohistochemistry was used to detect integumental MRCs in the yolk-sac larvae tail of the Nile tilapia following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt. Mean active MRC volume was always significantly larger and displayed a greater staining intensity (GLM; P<0.05) than nonactive MRCs. Following transfer, the percentage of active MRCs was seen to increase as did MRC volume (GLM; P<0.05). Microsc. Res. Tech., 2013. © 2013 Wiley Periodicals, Inc.