Imaging technologies have developed over the past decade in numerous ways to image sub-cellular structures with high resolution. Previously, fluorescence microscopy was constrained by the diffraction limit, a physical principle which states that light emitting species cannot be distinguishable when separated by small radii on the order of the wavelength of light used for imaging. Single-molecule localization microscopy (SMLM) microscopy is a new method which employs photoswitching fluorophores as the fluorescent labels. These are small organic dyes which switch stochastically between a light-emitting on-state and a non-emitting off-state. A sample is heavily labeled using these fluorescent dyes, and numerous photographic images are taken over the course of their blinking cycles. Since each image only has a fraction of molecules in the on-state, the radius between any given pair is significantly increased, and single emitters can be isolated and analyzed circumventing the diffraction problem. The result is a compiled image of all single frames, which exhibits near nanometer resolution. In order for this method to work, the fluorophores used must behave ideally with specific desirable blinking patterns. Thus, it is essential for developers in this field to have access to kinetics information and statistical models of the blinking behaviors of various commercial dyes. Here, a kinetics analysis is performed on fluorescent dye AlexaFluor 647 by taking fluorescence microscope single molecule imaging data and evaluating it using Microsoft Excel and ImageJ. The results indicate that the dye molecules perform better when immersed in a fluorescence-quenching buffer solution, in that their blinking patterns are more stable. Additionally, the methods herein provide a potentially standard approach to dye analysis using easily accessible, user-friendly software.

Kinetics study of Alexa Fluor 647 for single-molecule microscopy

ACS Spring Meeting 2021

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