The fidgety yeast: focus on high-resolution live yeast cell microscopy.
Despite its small size of 5-8 mum - only one order of magnitude above the wavelength of visible light - yeast has developed into an attractive system for light microscopic analysis. First, the ease of genetic manipulation and integrative transformation have opened numerous experimental strategies for genome-wide tagging approaches, e.g., with fluorescent proteins (as discussed in several chapters of this issue). Second, the large number of cells that can be simultaneously visualized provides an excellent basis for statistical image analysis, resulting in reliable morphological or localization information. Third, the flexibility of yeast cultivation in terms of biochemical manipulation, rapid cellular growth, mutant isolation or drug susceptibility offers an unprecedented spectrum of possibilities for in vivo functional studies, and analysis of cellular dynamics and organelle inheritance. Although yeast in itself is an interesting cellular system, its "prototype character" in understanding cellular metabolism, physiology, and signaling in eukaryotes accounts for its popular use in technology development and biomedical research.Here we discuss experimental strategies for live yeast cell imaging, geared towards imaging-based large-scale screens. Major emphasis is on the methods for immobilizing cells under "physiological" conditions, with minimum impact on yeast. We also point out potential pitfalls resulting from live cell imaging that once again stresses the necessity for extremely careful experimental design and interpretation of data resulting from imaging experiments. It goes without saying that these problems are not restricted to yeast and are also highly relevant to "large" cells. If an image tells more than a thousand (perhaps misleading?) words, the ease of obtaining "images" thus rather suggests analyzing many thousands of images, to come up with one relevant and biologically significant conclusion.
Wolinski
Heimo
H
Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50/II, A8010, Graz, Austria.
Natter
Klaus
K
Kohlwein
Sepp D
SD
D001483:Base Sequence; D018914:Cells, Immobilized; D017931:DNA Primers / Q000235:genetics; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007834:Lasers; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008857:Microscopy, Interference / Q000379:methods; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins