A fundamental component of quantitative cell biology is the ability to count molecules within cells. The numbers of molecules and stoichiometries are the basis for structural models of protein complexes and simulations of biological processes. A variety of methods exist for in vivo quantifications, but the focus of this volume is mainly on fluorescence methods. The two most popular methods are stepwise photobleaching and ratio comparison using a standard curve. With recent advances in genome editing techniques, most model organisms are amenable to inserting coding sequences for fluorescent proteins into native genetic loci, making quantification of proteins by fluorescence microscopy one of the most ubiquitous tools available to cell biologists.
The acquisition and analysis methods range from simple to complex, and most have been validated by counting with multiple methods and other types of data. Researchers should be aware of sources of error in the acquisition and analysis, but the accuracy of these methods is high. Quantification by fluorescence microscopy has yielded valuable new insights into many aspects of cell biology, highlighting its place among the standard tools for molecular and cell biologists.
Table of Contents
Recent Insights in Cell Biology
Counting Molecules by Fluorescence Microscopy
Variation and Sources of Error
Comparison with Complementary Approaches
About the Author(s)Valerie Coffman
, Department of Molecular Genetics, The Ohio State University, Columbus, Ohio
Valerie C. Coffman, Ph.D., is a postdoctoral researcher in the laboratory of Dr. Adriana T. Dawes (Mathematics, The Ohio State University). She received her Ph.D. in Molecular Genetics in 2013 from The Ohio State University, where she studied the involvement of formins in fission yeast cytokinesis with Dr. Jian-Qiu Wu. She received a predoctoral fellowship from the American Heart Association Great Rivers Affiliate, and an internal Elizabeth Clay Howald Presidential Fellowship, one of the highest honors bestowed by the graduate school to Ph.D. candidates in the final year of study. She is currently supported by a two-year Pelotonia postdoctoral fellowship. Three of her first-author publications in the Wu lab were highlighted by the journals as research of particular interest to the cell biology community. She has been a member of the American Society for Cell Biology since 2011.I-Ju Lee
, Department of Molecular Genetics, The Ohio State University, Columbus, Ohio Graduate Program of Molecular, Cellular, and Development Biology, The Ohio State University, Columbus, Ohio
I-Ju Lee, Ph.D., is a postdoctoral researcher at the Dana-Farber Cancer Institute, Harvard Medical School. She received her Ph.D. in Molecular, Cellular, and Developmental Biology in 2013 from The Ohio State University, advised by Dr. Jian-Qiu Wu. She studied the assembly of the contractile ring and spindle pole body. She received a two-year Pelotonia predoctoral fellowship in 2012. She has several first author publications, and two of her co-authored publications were highlighted by the journals as research of particular interest to the field. She has been selected to give oral presentations at several symposia, and received two poster awards for her work on nematodes and fission yeast. Jian-Qiu Wu
, Department of Molecular Genetics, The Ohio State University, Columbus, Ohio Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio
Jian-Qiu Wu, Ph.D., is an Associate Professor in Molecular Genetics at The Ohio State University. He received his Ph.D. in Biology in 2001 from The University of North Carolina at Chapel Hill, where he studied under Dr. John R. Pringle. He did his postdoctoral research with Dr. Thomas D. Pollard at Yale University, where he first counted protein molecules using fluorescence microscopy and quantitative immunoblotting. He also collaborated on a biophysical model of contractile-ring assembly in fission yeast partly based on the counted protein stoichiometries. His lab has been funded by an American Heart Association Beginning Grant-in-Aid, a March of Dimes Basil Oâ€™Connor Starter Scholar Research Award, an American Cancer Society Research Scholar Grant, and a National Institutes of Health R01 grant. He has been a member of the American Society for Cell Biology since 2001 and a member of the editorial board of BioArchitecture since 2012.