Self-Blinking
Dyes Unlock High-Order and Multiplane
Super-Resolution Optical Fluctuation Imaging
Posted on 2020-06-26 - 16:33
Most
diffraction-unlimited super-resolution imaging critically
depends on the switching of fluorophores between at least two states,
often induced using intense laser light and specialized buffers or
UV radiation. Recently, so-called self-blinking dyes that switch spontaneously
between an open, fluorescent “on” state and a closed,
colorless “off” state were introduced. Here, we exploit
the synergy between super-resolution optical fluctuation imaging (SOFI)
and spontaneously switching fluorophores for 2D and 3D imaging. SOFI
analyzes higher order statistics of fluctuations in the fluorophore
emission instead of localizing individual molecules. It thereby tolerates
a broad range of labeling densities, switching behavior, and probe
brightness. Thus, even dyes that exhibit spontaneous blinking characteristics
that are not suitable or suboptimal for single molecule localization
microscopy can be used successfully for SOFI-based super-resolution
imaging. We demonstrate 2D imaging of fixed cells with almost uniform
resolution up to 50–60 nm in 6th order SOFI and characterize
changing experimental conditions. Next, we investigate volumetric
imaging using biplane and eight-plane data acquisition. We extend
3D cross-cumulant analysis to 4th order, achieving super-resolution
in 3D with up to 29 depth planes. Finally, the low laser excitation
intensities needed for single and biplane self-blinking SOFI are well
suited for live-cell imaging. We show the perspective for time-resolved
imaging by observing slow membrane movements in cells. Self-blinking
SOFI thus provides a more robust alternative route for easy-to-use
2D and 3D high-resolution imaging.
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