Extended Data Fig. 5: FRET in HeLa cells expressing SYFP2 alone or SYFP2-RFP fusion proteins.
From: mScarlet3: a brilliant and fast-maturing red fluorescent protein
a. Average FRET spectra from HeLa cells expressing SYFP2 alone or SYFP2-RFP fusion proteins. All spectra were normalized to the unquenched SYFP2 donor. b. Quantification of FRET spectra from a. E (blue bars) represent the energy transfer efficiency (average ± sd), and Qs (red bars) depict the sensitized emission quantum yield (average ± sd). Qs is defined as the number of sensitized emission quanta emitted by the RFP acceptor divided by the number of quanta absorbed by the SYFP2 donor and is calculated by Qs=E·Qa, in which Qa is the acceptor quantum yield of fluorescence. Qs is not dependent on the emission spectra and is proportional to the integrated acceptor sensitized emission signal. Each data point represents quantitative unmixing analysis of a single cell spectrum, 18–25 single cell spectra were recorded for each condition. c. SYFP2 fluorescence lifetime of HeLa cells expressing SYFP2 alone or SYFP2-RFP fusion proteins as determined by frequency-domain FLIM. Tau(phi) (red bars) is the average lifetime estimated from the phase shift (± sd), Tau(mod) (blue bars) is the average fluorescence lifetime estimated from the demodulation (± sd). For c each data point represents the average lifetime of an image with 4–20 individual cells, 4–6 FLIM recordings were done for each condition.
