Photobleaching

Photobleaching is an unavoidable phenomenon in fluorescent microscopy that occurs when a fluorophore in a fluorescently labeled specimen forms a covalent bond with another molecule. As a result the fluorophore can no longer decay to its original state and emit a photon. In this lab we tested for the rate of photobleaching under various conditions and with three types of fluorescent stains: DAPI, fluorescein, and rhodamine. The intensity of nuclei were calculated over varying exposure times and were analyzed. We concluded that the use of an automatic shutter and excitation filters greatly reduced the rate of photobleaching for all fluorescent stains. Our data also suggested that DAPI photobleached at the lowest rate compared to the other fluorophores. Overall, fluorescent microscopy is an efficient way to visualize specific targets given that proper precautions are taken to prevent photobleaching.