Imaging Analysis of Photoswitching Fluorophores Using Single-Molecule Microscopy

Single-molecule localization microscopy (SMLM) is a developing field of biological imaging that employs the use of photoswitching fluorophores to image sub-cellular biological structures at a higher resolution than was previously possible. These fluorophores are used for protein labeling, so that the sample can be imaged under fluorescence microscopy. This type of microscopy requires the use of many different types of fluorophores, which are fluorescent organic compounds that blink stochastically on and off. Thus, it is critical for developers in the field to have easy access to statistical models of the behaviors of different fluorophores. Here, we take AlexaFluor 647 and analyze it using a fluorescence microscope, taking data on its blinking behaviors and discerning its properties when immersed in a fluorescence-dampening buffer solution. We find that the compound behaves best in buffer solution, and we forge a new methodology for evaluating new fluorophores in a systematic fashion using readily available computer software