TY - JOUR
T1 - Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation
AU - Hodgson, Louis
AU - Nalbant, Perihan
AU - Shen, Feimo
AU - Hahn, Klaus
N1 - Funding Information:
This work was supported by The National Institutes of Health (GM057464 and GM064346).
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
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U2 - 10.1016/S0076-6879(06)06012-5
DO - 10.1016/S0076-6879(06)06012-5
M3 - Review article
C2 - 16472656
AN - SCOPUS:32144456185
SN - 0076-6879
VL - 406
SP - 140
EP - 156
JO - Methods in enzymology
JF - Methods in enzymology
M1 - 12
ER -