Determining the optimal acquisition settings for FRET imaging can be tricky. The exposure settings for a CCD camera (or gain settings for a photomultiplier tube) must be identical for each channel (CFP, YFP, and FRET). This can be problematic, as YFP is often brighter than CFP under the same exposure settings. Thus, it is difficult to optimize the exposure of CFP without saturating the YFP signal. Therefore, we typically set the exposure settings using the YFP channel such that both YFP and CFP images have a signal-to-noise ratio that is at least 2:1. Because oxygen radicals are produced with high-intensity light, phototoxicity can be minimized by choosing longer exposures with dim illumination (provided by neutral density filters). However, exposures for live-cell FRET imaging should be as short as possible to minimize artifacts introduced by the movement of protein complexes during image acquisition. In cases of dim fluorescence, the recorded signal can often be increased by increasing the detector gain, binning data (for CCD detectors), or increasing the scan time (for confocal microscopes). Once acquisition parameters have been determined, they should not be changed during the course of the experiment.
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