Single-cell quantification of molecules and rates using open-source microscope-based cytometry

ANDREW GORDON (CO-PRIMER AUTOR); ALEJANDRO COLMAN-LERNER (CO-PRIMER AUTOR); TINA CHIN; KIRSTEN BENJAMIN; RICHARD YU; ROGER BRENT

Nature Methods

Nature Publishing Group

Lugar: New York, New York, USA; Año: 2007 vol. 4 p. 175 - 181

1548-7091

Microscope-based cytometry provides a powerful means to study cells in high throughput. Here we present a set of refined methods for making sensitive measurements of large numbers of individual Saccharomyces cerevisiae cells over time. The set consists of relatively simple ´wet´ methods, microscope procedures, open-source software tools and statistical routines. This combination is very sensitive, allowing detection and measurement of fewer than 350 fluorescent protein molecules per living yeast cell. These methods enabled new protocols, including ´snapshot´ protocols to calculate rates of maturation and degradation of molecular species, including a GFP derivative and a native mRNA, in unperturbed, exponentially growing yeast cells. Owing to their sensitivity, accuracy and ability to track changes in individual cells over time, these microscope methods may complement flow-cytometric measurements for studies of the quantitative physiology of cellular systems.