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Fluorescence [excitation 485 (20-nm bandwidth), emission 530 nm (25-nm bandwidth)] was measured using a Synergy2 multifunction plate reader (BioTek, Winooski, VT).
The following parameters were employed: spectral bandwidth 1 nm, step size 0.5 nm and time-per-point 0.5 s.
Spectral bandwidth: 5 nm; step size: 1 nm; scan time: 120 s.
The excitation wavelength (bandwidth 5 nm) was set to 330 nm to avoid the Raman peak of water.
The fluorescence emission (bandwidth 2 nm) was measured from 365 to 600 nm in 0.5-nm increments.
Bandpass filters (bandwidth 0.5 nm) are used in both arms to minimise the white noise from the EDFAs.
For FRET imaging, CFP was excited by a narrow bandwidth 436/10 nm filter to minimize direct excitation of fura-2.
The pathlength was 10 mm, spectral bandwidth 1 nm and scan time per point was set at 10 s.
The pathlength was at 1 mm, spectral bandwidth 3 nm and scan time per point was set at 10 s.
The pathlength was 10 mm, spectral bandwidth 1 nm and the scan time per point was set at 10 s.
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