User: Diane Lidke, University of New Mexico School of Medicine, Albuquerque
The project: Labeling as many as six proteins simultaneously to track protein dynamics during signal transduction in live cells.
The problem: Lidke chooses quantum dots (Invitrogen) over dyes "mainly for their photostability and high brightness." However, even quantum dots can degrade with high excitation power. Light degradation "will result in a loss of fluorescence intensity," Lidke says, "but before all fluorescence is lost, the emission wavelength will slowly shift to the blue," potentially causing dot emission to be collected in the wrong detection channel. Moreover, methanol and mounting media can damage quantum dot fluorescence.
The solution: The only solution to light degradation is to decrease excitation power, Lidke says, either by attenuating the laser in confocal microscopy or by using a neutral-density filter in front of the lamp in wide-field microscopy. "In general, one does not need to image ...
