Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.
This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.
Department of Pharmacology, University of North Carolina, Chapel Hill, USA.
Louis
L
Hodgson
Perihan
P
Nalbant
Feimo
F
Shen
Klaus
K
Hahn
D015374:Biosensing Techniques / Q000379:methods*; D002465:Cell Movement; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D011744:Pyrimidinones / Q000737:chemistry; D012984:Software; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011744:Pyrimidinones; D051316:Wiskott-Aldrich Syndrome Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C003954:merocyanine dye; D020764:cdc42 GTP-Binding Protein