What can cause artifacts in fluorescence imaging of GFP?

Photobleaching or overexpression of GFP can lead to artifacts in fluorescence imaging for several reasons.

Photobleaching occurs when the fluorophore (in this case, GFP) loses its ability to fluoresce due to prolonged exposure to excitation light. This can lead to an uneven distribution of fluorescence intensity across the sample, as some areas may appear dimmer or lack fluorescence altogether due to the degradation of the GFP molecules. This effect can confuse the interpretation of the data, leading to misrepresentation of the protein localization and activity.

Overexpression of GFP can result in excessive amounts of the fluorescent protein in specific areas of the cell, causing fluorescence signals to saturate. When the fluorescence signal saturates, it may not represent a true quantitative measure of the protein's localization or concentration, leading to misleading conclusions from the imaging assays. This saturation can create artifacts such as clustering effects, where the signal appears much stronger than it actually is due to the concentration of the protein in small areas.

Both photobleaching and overexpression highlight the importance of optimizing conditions in fluorescence imaging to obtain accurate and meaningful results, making B the correct choice regarding factors that can cause artifacts in GFP fluorescence imaging.