Single-molecule localization microscopy (SMLM) is an imaging method which utilizes the stochastic blinking characteristics of photoswitching fluorophores to obtain images with higher resolution than the diffraction limit would allow with traditional microscopy. Photoswitching fluorophores, compounds which on the single molecule level stochastically fluoresce, exhibit a variety of kinetic characteristics which determine statistics of blinking and reaction rates, that must be accurately modeled for use in SMLM. Because of this, it is necessary to accurately and repeatably quantify each fluorophore’s kinetic properties with a systematic method. Sequential images obtained via fluorescence microscopy of AlexaFluor 647 beads were analyzed with the application FIJI. The raw data from FIJI was further analyzed with a custom python script, which interacted with Microsoft Excel in order to obtain average on-times, duty cycles, and rate constants. The results were compared to a previous study in order to determine the accuracy of this computational method.

Computational processing of photoswitching fluorophore blinking kinetics for single-molecule microscopy

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.