New stereoscopic reconstruction protocol for scanning electron microscope images and its application to in vivo replicas of the shoot apical meristem

Abstract

The shoot apical meristem is a small and delicate organ, usually hidden between the young leaves and flowers that it produces. One approach to study meristem geometry and growth consists of taking consecutive replicas from the living meristem surface. In this paper, we present a new stereoscopic reconstruction method for this non-invasive replica protocol, which is applicable to study of growth and geometry of individual cells. This method had been used by the authors to study shoot apical meristem of two species: Arabidopsis thaliana (L.) Heynh. and Anagallis arvensis L., and can be extended to other species and organs. Scanning electron micrographs of the same replica are made at two different angles of view. The obtained stereopairs are used for the dense, three dimensional reconstruction of the replica surface. At the same time, some of the microscope parameters are refined based on the differences between the two micrographs. Three dimensional cell outlines are next extracted from the dense continuous reconstruction, and provide a basis for the quantification of meristem geometry and growth. The new reconstruction protocol can be used with different types of scanning electron microscopes, single- or multi-staged, does not require the identical working distance for the two micrographs of the stereopair, and can be used within a large range of magnifications, corresponding to the cases of either orthogonal or central projection model. It is based largely on recently published algorithms for stereoscopic vision. The reconstruction protocol can be used also for other stereoscopic applications based on scanning electron microscopy. The codes are written in Matlab and are freely available on request to the authors.