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

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Superresolution microscopy relies on photoswitchable dyes, often requiring enzymatic oxygen scavengers.
• Enzymatic oxygen scavengers interfere with atomic force microscopy (AFM) cantilevers, hindering correlative imaging.
• Existing red dyes like Alexa-647 have limitations in performance and compatibility with AFM.

Purpose of the Study:

• To evaluate the blinking kinetics of a novel red cyanine dye, iFluor-647.
• To compare iFluor-647 performance against Alexa-647 in buffers with and without enzymatic oxygen scavengers.
• To demonstrate the utility of iFluor-647 for correlative superresolution and AFM without buffer exchange.

Main Methods:

• Blinking kinetics measurements of iFluor-647 in various buffer conditions (with/without oxygen scavenger, varying thiol concentrations).
• Characterization of iFluor-647's photophysical properties.
• Application of iFluor-647 in a correlative superresolution and AFM workflow.

Main Results:

• iFluor-647 exhibits superior brightness and blinking properties compared to Alexa-647 in a buffer lacking enzymatic oxygen scavenger.
• The dye's blinking behavior is characterized across different thiol concentrations and the presence/absence of oxygen scavengers.
• Successful correlative superresolution and AFM imaging was achieved using iFluor-647 in a buffer free of enzymatic oxygen scavenger.

Conclusions:

• iFluor-647 is a promising alternative red dye for superresolution microscopy.
• The dye's performance in non-scavenging buffers facilitates seamless correlative superresolution and AFM.
• This work overcomes a key limitation in correlative microscopy, enabling simultaneous acquisition of optical and topographical data.