Spinning-disk confocal microscopy of yeast.
Spinning-disk confocal microscopy is an imaging technique that combines the out-of-focus light rejection of confocal microscopy with the high sensitivity of wide-field microscopy. Because of its unique features, it is well suited to high-resolution imaging of yeast and other small cells. Elimination of out-of-focus light significantly improves the image contrast and signal-to-noise ratio, making it easier to resolve and quantitate small, dim structures in the cell. These features make spinning-disk confocal microscopy an excellent technique for studying protein localization and dynamics in yeast. In this review, I describe the rationale behind using spinning-disk confocal imaging for yeast, hardware considerations when assembling a spinning-disk confocal scope, and methods for strain preparation and imaging. In particular, I discuss choices of objective lens and camera, choice of fluorescent proteins for tagging yeast genes, and methods for sample preparation.
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California, USA.
Kurt
K
Thorn
D005656:Fungal Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D015003:Yeasts / Q000378:metabolism*
D005656:Fungal Proteins