We present here a protocol that involves genetically coupled spectrally distinct photo-activatable and fluorescent proteins.These internal rulers permit quantification of the PAFP fraction that is photo activated to be fluorescent.Thus far, no method has been available to quantify in bulk in life cells how many of the PAFP expressed are photo activated to fluoresce.The presented quantification of photo-activation efficiency is exemplified for PA-GFP and PA Cherry in live cells but it is in principle broadly applicable and can be used for any photo-activatable fluorescent protein under any experimental condition.Whenever working with genetically encoded fluorescent proteins, the absolute amount of proteins expressed varies from cell to cell and it is unknown.To standardize the expression among cells, we introduce internal rulers of genetically coupled spectrally distinct fluorescent proteins.By coupling the genetic information of a photo-activatable florescent protein to a spectrally distinct always on fluorescent protein internal rulers are created that will still be expressed to an unknown total amount but in a fixed known relative amount of one to one.To begin construct the plasmids as described in the accompanying text protocol.Also, culture in the standard cell line in its perspective medium using the phenol red free version.When ready to begin the experiment, harvest the cells using trypsin without phenol red to reduce background florescence.After harvest, fill a 2 ml pipette with the cell-suspension from a confluent cell culture grown in a T 12.5 cell culture flask.Add one drop into each chamber of an eight chamber glass slide.Incubate the slide under standard cell culture conditions for 24 hours.Next transfect the cells using commercial reagents following the distributors protocol.Transfect the cells with the GFP Cherry, PA-GFP Cherry and GFPPA Cherry chimeras.Then return the cells to the incubator to allow for protein expression, folding, and maturation.After a total of 20 hours post transfection, transfer the cells to the stage of a confocal florescent microscope equipped with the humidified and heated environmental chamber pre-warmed to 37
