Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

TLDR

Super‑resolution optical microscopy is rapidly evolving and can surpass the diffraction limit, impacting many scientific fields. The authors present an approach to achieve sub‑diffraction optical resolution in all three dimensions. The method relies on higher‑order statistical analysis of fluorescence blinking recorded in a movie, applied iteratively in discrete steps to evaluate images at multiple resolution levels. The technique yields a five‑fold spatial resolution improvement on a conventional wide‑field microscope and, even at the lowest enhancement level, delivers substantial background reduction and contrast enhancement on quantum‑dot‑labeled microtubules.

Abstract

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.