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.
Thorn
Kurt
K
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California, USA.
D005656:Fungal Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D015003:Yeasts / Q000378:metabolism*
D005656:Fungal Proteins