Uv Light Induced Fluorescence Recovery of GFP After Photobleaching in Microscopy Imaging

Abstract

Fluorescence microscopy has become one of the most important tools for biologists to visualize and study organelles and molecules in a cell. Fluorescent markers are used to visualize specific molecules. One of the most used markers is green fluorescent protein (GFP), which can be expressed along with a protein of interest. However, it is known that the intensity of fluorescence decreases with observation time. To combat this problem, researchers and companies have developed protocols and additives to mitigate photobleaching. In this study, we tested the effects of the three most used culture media on photobleaching and developed a new approach of short-wavelength fluorescence recovery after photobleaching (FRAP). Photobleaching was analyzed by comparing pixel brightness on images taken with a fluorescence microscope. We determined photobleaching of GFP-expressing cells from images taken with a fluorescence microscope by comparing pixel brightness. Statistical analysis was performed to determine the average bleaching for specific culture media. The culture media analyzed had no significant effect on the photobleaching of GFP. However, a brief UV burst (15 sec) restores 50% of the original fluorescence and neither increases ROS nor decreases cell viability. To avoid artifacts in image analysis and interpretation, our study suggests using this simple method of GFP fluorescence recovery with UV light induced FRAP to extend the fluorescence lifetime and imaging of GFP molecules.

Enhanced fluorescence blinking of AF647 fluorophores in Mowiol via violet and UV light induced recovery for superior localization microscopy

“…The formation of the long-lived dark states (R) is so prominent that after a few seconds under an intense excitation beam, the molecules stop taking part in the continuous fluorescence emission process as they move to the R state [27]. On the other hand at low excitation intensity, a camera with milli-second exposure time can rarely detect any fluorescence blinking and there is primarily continuous fluorescence emission [28]. Therefore increasing the excitation intensity from low to high is important to switch from negligible blinking to observable blinking [22].…”