Fig. 2. The method of 3D micromorphometrics. (A) In this set-up the larva is positioned beneath the water-immersion objective of a compound microscope. The microscope is free to translate in three dimensions to interrogate microscopic features within the specimen. (i–iv) The position of these features is measured with the aid of a custom-designed computer program that first (i) captures digital photographs of the microscope field of view. Each photograph captures morphology at a particular optical plane with the z-position determined by the microscope focus. (ii) The user selects landmarks from this image. Once the user has entered the position of the microscope objective in global coordinates, (iv) the 3D positions of landmarks are calculated (see Materials and methods for details). (B) These coordinates are described with respect to the central axis of the body (dashed line), which is defined by points at the rostrum and tail tip. (C) The seven landmarks from a neuromast were used to calculate the cupular height (h_c), kinocilia height (h_k), base diameter (d_b) and diameter at the kinocilia height (d_k).