Live Cell Imaging of Yeast

INTRODUCTION

The development of cloning vectors for green fluorescent protein (GFP) and the simplicity of yeast reverse genetics allow straightforward labeling of yeast proteins in living cells. Budding and fission yeast are therefore attractive organisms in which to study dynamic cellular processes such as growth, cell division, and morphogenesis using live cell fluorescence microscopy. This article focuses on methods to culture, mount, and observe budding yeast cells using three-dimensional (3D) microscopy, but the methods are broadly applicable to other types of cells and other imaging techniques. The emphasis is on 3D imaging, because yeast cells are roughly spherical, and most organelles in yeast move in three dimensions. Three-dimensional imaging also makes it possible to apply image restoration methods (e.g., deconvolution) to obtain sharper images with better definition. This is important, because yeast cells are small (haploid Saccharomyces cerevisiae cells have a diameter of ~4–5 µm) relative to the resolution of even the best optical microscope (~0.25 µm).