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Methods Enzymol. 2006;406:140-56.

Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.

Author information

1
Department of Pharmacology, University of North Carolina, Chapel Hill, USA.

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.

PMID:
16472656
DOI:
10.1016/S0076-6879(06)06012-5
[Indexed for MEDLINE]

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