STORM without enzymatic oxygen scavenging for correlative atomic force and fluorescence superresolution microscopy

Abstract

Superresolution microscopy based on localisation is usually performed in a buffer containing enzymatic oxygen scavenger, which facilitates reversible photoswitching of the dye molecules. This makes correlative fluorescence localisation and atomic force microscopy (AFM) challenging, because enzymatic oxygen scavenging interferes with the AFM cantilevers. Here we report on the blinking kinetics of a new red cyanine dye, iFluor-647, which is similar to the Alexa-647 dye commonly used for superresolution microscopy, but with brightness and blinking properties which are superior to Alexa-647 in a buffer without enzymatic oxygen scavenger. We measure the blinking behaviour of iFluor-647 in buffers with and without enzymatic oxygen scavenger with different thiol concentrations. We then apply this dye for correlative localisation and atomic force microscopy in a buffer without enzymatic oxygen scavenger, which allows acquisition of AFM and superresolution images without buffer change.