Abstract
The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report the construction, properties, and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.
Lay Abstract A broad goal of modern biology is to understand how the machines within living cells work. It is nowadays routine to identify the individual protein components of a machine, but not yet straightforward to tell how many copies of each component are needed to build a functional assembly. In many types of cells it is now possible to substitute for the native proteins within cells altered versions that are fluorescent. If one knew how much fluorescence is generated by a single molecule of the altered protein, then one could use a light microscope to count the number of copies of the protein in a cellular machine by simply measuring the total fluorescence coming from that part of the cell. This paper describes the construction and methods for using a set of fluorescent virus particles that can be used to determine how much fluorescence is contributed by one molecule of fluorescent protein. The virus particles were chosen for this role because the particular icosahedral symmetry of their structure guarantees that each particle contains exactly 240 copies of one fluorescent protein.
