Simple buffers for 3D STORM microscopy

Abstract

3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30–50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

Advances in Imaging Plant Cell Dynamics

“…Moreover, the blinking of fluorophores suitable for STORM requires special buffer conditions, but with the appropriate fluorophore-buffer combination, the resolution can reach 5 nm (Dempsey et al, 2011). Efficient blinking of commonly used STORM fluorophores can be achieved in commercial mounting media, especially in Vectashield (Olivier et al, 2013). The introduction of Escherichia coli dihydrofolate reductase (eDHFR) as a protein tag of intracellular structures allows the labeling of eDHFR fusion proteins with fluorophore-conjugated trimethoprim primers used for live STORM applications (Wombacher et al, 2010).…”

Twinkle, twinkle little star: Photoswitchable fluorophores for super‐resolution imaging

“…The original STORM/PALM publication with Cy5 used an imaging cocktail containing an oxygen scavenger system and thiol to facilitate photoswitching [15]. Subsequently, it has been shown that Cy5 may be: switched on and off using redox chemistry with ascorbic acid [36]; reduced with NaBH 4 to a long-lived non-fluorescent form and photoactivated [32]; switched on and off using the phosphine TCEP (tris 2-carboxyethly phosphine) [37]; switched on and off using a thiol and a mixture of either cyclooctatetraene or unknown additives in the proprietary sample mounting medium Vectashield [38,39]. For these different schemes, photons detected per localization varied between $100-1 000 000, and duty cycle, which is generally harder to measure, in the neighborhood of 10 À2 to 10 À6 .…”

Assessing resolution in super-resolution imaging

“…After a large number of repeats, 1000-100,000 or more, a large number of individual fluorophore positions can be acquired and a super-resolution image produced. Initial efforts using this technique focused on using photo-switching mechanisms to turn on and off individual molecules, but the field has largely moved on to using high illumination intensities (10-50 kW/cm 2 ), sometimes in combination with specific buffers [26], and relying on transferring a large percentage of molecules into a long-lived dark state. As they spontaneously return to a fluorescent state they are imaged, and then bleached or returned to a dark state.…”