FRET Image analysis-Spillover factors

The next step is to select an appropriate 5 x 5 pixel background box. The choice of a background region is a critical step. In the case of the SPB we first examine the DIC image, below, to orient the position of the selection box. The background region will be an adjacent square within the cell, as shown in the next web page. If the protein had a diffuse nuclear pattern, an adjacent region in the cytoplasm would be chosen. The most challenging case is a signal that is present throughout the cell. In that case we mix in isogenic cells without a label and use a region in an adjacent cell as background. The choice of background requires your best judgement. One check on the choice of background is that a negative control for FRET will have a FRETR value different from the expected value of 1.00.