Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation
In: Methods in Enzymology, Vol. 406 (2006), No. 12, pp. 140 - 156
Title:
Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation
Author:
Hodgson, Louis;Nalbant, PerihanUDE
- LSF ID
- 49819
- ORCID
- 0000-0002-5649-5933
- Other
- connected with university
Year of publication:
2006
Abstract:
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.