In this research project, we have so far obtained the following findings. First, Nural tube closure (NTC) of ascidian is a good experimental model to observe because of relatively short period of NTC and simple body plan consists of small number of cells. To understand ascidian NTC at single cell level, nucleus of neuronal cells were labeled and were observed by 4D (3D timelapse) imaging throughout NTC process. Observed nucleus was digitally tracked and the cell lineage was traced. From this quantitative analysis, it was revealed various kinds of specific behaviors of cell including cell division in specific regions of ascidian brain (unpublished data). Second, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm(-1), respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis (Nakamura et al., 2013 PlosOne).