Researchers studying proteins in heart cells have unexpectedly discovered that a common microscopy fluorescent protein carries reversible photobleaching properties. This phenomenon may lead to inaccurate or unreliable data.
The study is published ahead of print in the American Journal of Physiology-;Cell Physiology and was chosen as an APSselect article for January.
Fluorescence recovery after photobleaching (FRAP) is a technique that uses fluorescent microscopy to determine the amount and rate of protein movement in a cell or region of a cell. Scientists using FRAP mark a part of a cell with a fluorescent protein tag and take time-lapse images to capture the cell's fluorescent protein signal.
Then, they use a high-power laser that removes the protein's fluorescence in a process called photobleaching. The final phase of FRAP is recovery: the cell begins to emit fluorescence again when new fluorescent proteins are produced or transported within the cell.
One important principle in FRAP is the assumption that photobleaching is irreversible, as the [laser] disrupts the fluorescence protein structure and function."
Adrian Cadar, PhD, first author of the study
Researchers performed FRAP-;using a fluorescent protein called mEos3.2-;on beating human heart cells to learn more about the turnover of titin. Titin is a very large, elastic protein that forms a complex molecular framework of muscle cells called the sarcomere.
Previous research has found that the sarcomere is a dynamic structure in which there is a constant and rapid exchange of titin molecules. However, in the current study, titin tagged with mEos3.2 had a much quicker recovery time than the research team had anticipated.
To determine if the recovery signal was indeed due to titin movement, the team performed a critical control by treating the cells with paraformaldehyde-;a process called fixation. The fixation process crosslinks the proteins within the cell together, thus immobilizing them.
The researchers then performed FRAP in these fixed cells, which surprisingly revealed a fluorescence recovery signal similar to the initial live cell measurements.
The researchers discovered that, unlike other proteins used in FRAP, mEos3.2 refolds or restructures itself to regain fluorescence. This reversibility leads to unreliable data.
"When using fluorescent proteins for FRAP studies, it is important that one validates the photo-properties of their particular fluorescent protein. In our case, the mEos3.2 photoconvertible fluorescent protein displayed a significant reversible photobleaching property which hasn't been described before. This property makes the mEos3.2 fluorescent protein an inappropriate tool for FRAP studies as the reversible photobleaching led to an overestimation of the fluorescent recovery signal," Cadar wrote.