Dynamic study of cellular events during cryopreservation
Abstract
Conventional microscopy techniques present some limits for studying cellular events occurring through the various steps of a cryopreservation process. It is particularly difficult to quantify and topologically identify cellular changes. These last years, new imaging techniques were developed, mainly in animal sciences, creating real time or 3D images. The Real-Time Microscopy (RTM) permit to observe living cells without any stain or contrast agent. Combined with a perfused disposable chamber it is possible to change the cellular environment during the observation. Confocal Laser Scanning Microscopy (CLSM), combined with special software, allows three-dimensional reconstructions of cells, tissues or organs. We tried to adapt these two techniques in order to better understand Pelargonium apex evolution during some steps of a droplet-vitrification protocol. The first step was to determine the degree of feasibility of the project. For practical reasons, we first focus on the structural modifications due to LS and PVS2 addition. We demonstrated that it is possible to have a sequence of the events in real time and to quantify them.