Abstract
Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial. Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope. SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.
Highlights
Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, remains a challenge as its implementation by existing superresolution methods is non-trivial
The sectioning, background suppression and resolution enhancement afforded by superresolution optical fluctuation imaging (SOFI) are apparent for the stack
The resolution enhancement along the optical axis is difficult to estimate, since the axial resolution of the original widefield data set is ill-defined
Summary
Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, remains a challenge as its implementation by existing superresolution methods is non-trivial. Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. Superresolution (SR) imaging has revolutionized fluorescence biological imaging by providing resolution enhancement down to a few 10’s of nanometers, allowing us to decipher morphology of small organelles and sub-cellular structures Superresolution methods, generically provide enhanced resolution in two dimensions (2D) only (Betzig et al 2006; Rittweger et al 2009; Rust et al 2006); additional resolution enhancement along the optical axis (i.e. SR in three dimensions, 3D) is a more challenging task and usually requires additional (and often significant) modifications to the optical set-up (Nagorni and Hell 2001; Pavani et al 2009; Roman et al 2008; Shtengel et al 2009). In single-molecule localization methods, the imaging depth that supports
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