Zebrafish is a model organism in developmental biology for several reasons, including the fact that Zebrafish embryos are translucent and this enables the study of tissue development under a microscope. This study is aimed at developing methodologies for characterizing the metabolic state of different tissues during development and in different controlled environmental conditions. Two-photon excited fluorescence (TPEF) microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were used to study different tissues during embryos development and to characterize them in various controlled environmental conditions. The extensive anatomical mapping of the Zebrafish embryos was obtained through the detection of autofluorescence. Differences in metabolism among different organs were observed by measuring NADH/FAD ratio and free NADH / bound NADH ratio. The proposed method has the potential for monitoring embryo metabolism in different conditions during development.