Besides molecular-biological, genomic and proteomic techniques, high-resolution light microscopy is important for gaining innovative insights into cellular lipid synthesis, lipid and membrane dynamics and morphological changes in connection with lipid metabolism disorders.
With its diameter of 5–8 μm, the yeast cell used to be considered an unsuitable specimen for light microscopic examination. Actually, the technological progress made in microscopy over the last few years through improved imaging techniques and excellent objectives can now resolve sub-cellular structures of yeast cells (Figure 1). Confocal laser scanning microscopy offers special advantages for three-dimensional analysis of cells observed under physiological conditions over several generations . Diffraction problems and stray light are minimal due to the thin cell thickness. The typical lateral and axial resolution is approx. 150 nm and 350 nm, respectively. The three-dimensional reconstruction achieved by recording a large number of “optical sections” and the simultaneous detection of several fluorophores provide completely new insights into the spatial and dynamic protein and lipid interactions in live cells.
The availability of fluorescing protein variants (e.g. GFP – green fluorescent protein) in connection with extremely simple cloning techniques has created the basis for localisation and expression studies of all proteins of the yeast proteome (of which there are roughly 6,000) . Robust preparation protocols for live cell microscopy and vital staining allow simultaneous observation of large cell populations, an excellent basis for collecting quantitative microscopy data.