Journal
BIOSENSORS & BIOELECTRONICS
Volume 46, Issue -, Pages 97-101Publisher
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2013.02.024
Keywords
Fluorescent protein; FRET biosensor; Multi-parameter imaging; mTagBFP; sfGFP; mVenus; mKO kappa
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Funding
- National Basic Research Program of China [2011CB910401]
- Science Fund for Creative Research Group of China [61121004]
- National Natural Science Foundation of China [81172153]
- National Science and Technology Support Program of China [2012BAI23B02]
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Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) biosensors are powerful tools for dynamically measuring cellular molecular events because they offer high spatial and temporal resolution in living cells. Despite the broad use of FP-based FRET biosensors in cell biology, imaging of multiple molecular events (multi-parameter molecular imaging) in single cells using current FRET pairs remains difficult because it usually requires a control group for additional data calibration. Hence, spectrally compatible FRET pairs that do not require complex image calibration are the key to widespread applications of FP-based FRET biosensors in multi-parameter molecular imaging. Here, we report a new combination of spectrally distinguishable FRET pairs for dual-parameter molecular imaging: mTagBFP/sfGFP (blue and green FP, B/G) and mVenus/mKO kappa (yellow and orange FP, Y/O). We demonstrate that additional image correction is not necessary for these dual FRET pairs. Using these dual FRET pairs, we achieve simultaneous imaging of Src and Ca2+ signaling in single living cells stimulated with epithelial growth factor (EGF). By converting traditional FRET biosensors into BIG and Y/O-based biosensors, additional applications are available to elucidate the dynamic relationships of multiple molecular events within a single living cell. (C) 2013 Elsevier B.V. All rights reserved.
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