I feel that the imaging itself is having an impact on our cell behavior and is skewing the experiment results. How would you set up a control test to verify? And if so, how can I minimize the impact on cell behavior?

Answer

This is a tricky question to answer. The experimental results in a fluorescence imaging experiment could be influenced by exposure to the fluorescent light, the imaging environment or the unnatural expression of an exogenous, fluorescently-labeled transgene. To control for the effect of imaging on cell behavior, it would be best to validate the findings of your imaging experiments with an alternative non-image-based cell or biochemical assay that doesn’t require exposure to fluorescent light or the expression of a transgene. If this is not possible, I would suggest the following:

1. Ensure that the imaging environment has the optimal CO2 concentration and temperature which are required for maintaining good cell health.

2. Use an imaging cell culture medium such as FluoroBrite that has very low background fluorescence but all the necessary nutrients for long-term cell health over hours or days. A low background medium will reduce the need for using high laser light intensities that can harm the cells or alter their behavior.

3. Perform a titration experiment where you expose the cells in your experimental system to a range of fluorescent light levels and/or exposure times and monitor the cell behavior over this range. This is still not ideal but could at least help you to identify the lowest amount of light exposure for imaging your cells, thereby minimizing any negative impact on cell behavior.