Methods to study organogenesis in decapod crustacean larvae II: analysing cells and tissues

Cells and tissues form the bewildering diversity of crustacean larval organ systems which are necessary for these organisms to autonomously survive in the plankton. For the developmental biologist, decapod crustaceans provide the fascinating opportunity to analyse how the adult organism unfolds from organ Anlagen compressed into a miniature larva in the sub-millimetre range. This publication is the second part of our survey of methods to study organogenesis in decapod crustacean larvae. In a companion paper, we have already described the techniques for culturing larvae in the laboratory and dissecting and chemically fixing their tissues for histological analyses. Here, we review various classical and more modern imaging techniques suitable for analyses of eidonomy, anatomy, and morphogenetic changes within decapod larval development, and protocols including many tips and tricks for successful research are provided. The methods cover reflected-light-based methods, autofluorescence-based imaging, scanning electron microscopy, usage of specific fluorescence markers, classical histology (paraffin, semithin and ultrathin sectioning combined with light and electron microscopy), X-ray microscopy (µCT), immunohistochemistry and usage of in vivo markers. For each method, we report our personal experience and give estimations of the method’s research possibilities, the effort needed, costs and provide an outlook for future directions of research.