Single-cell quantification of molecules and rates using open-source microscope-based cytometry.
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.
Gordon
Andrew
A
The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, California 94704, USA.
Colman-Lerner
Alejandro
A
Chin
Tina E
TE
Benjamin
Kirsten R
KR
Yu
Richard C
RC
Brent
Roger
R
D005434:Flow Cytometry; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis; D018922:HL-60 Cells; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D020871:RNA Stability; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000166:cytology / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors
D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins