Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Confocalists

One of the users of our facility has prepared cells in which he stained a surface protein by using an external epitope tag and a red labeled secondary, he also stained the same protein using an internal epitope and a green labeled secondary in order to asses the total level of expression per cell.

He wants to show the level of surface expression relative to the total cell expression of that protein. First he tried to approach this just by showing representative images of the different treatment conditions, of course the reviewers of his manuscript asked him for quantification in the form a ratio of surface to total protein expression.

I told him that he must be aware that although it is easy to calculate a ratio of the red intensity to the green intensity, the resulting value would be just proportional to the absolute ratio of surface expression and could only be used to compare the relative changes among the different treatments, but he would have a lot of variability due to uncontrolled variation in illumination sources, etc etc....

Of course trying to segment the green image in peripheral (near and at the surface) and total fluorescence would not be a solution either, due to the obvious limitations in the optical resolution.

I would appreciate any comments in that sense and if a relative ratio of red to green fluorescence would be in anyway acceptable to answer the reviewer.

Thanks in advance

Leoncio A. Vergara MD
Assistant Professor
Laboratory of Protein Misfolding Diseases (lab-PMD),
George and Cynthia Mitchell Center for Neurodegenerative Diseases Research.
Director of the Optical Imaging Lab. (OIL),
Dept. of Neuroscience and Cell Biology
University of Texas Medical Branch (UTMB)
301 University Blvd
Galveston, Texas 77555-0641
OIL phone: 409-772-3970
Lab-PMD phone: 409-7470019
fax: 409-7470015