Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).
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.
Dertinger
T
T
Department of Chemistry and Biochemistry, and California NanoSystems Institute, University of California, Los Angeles, CA 90095, USA. t.dertinger@chem.ucla.edu
Colyer
R
R
Iyer
G
G
Weiss
S
S
Enderlein
J
J
D016475:3T3 Cells; D000818:Animals; D055592:Biophysical Phenomena; D005347:Fibroblasts / Q000648:ultrastructure; D005456:Fluorescent Dyes; D021621:Imaging, Three-Dimensional; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D008870:Microtubules / Q000648:ultrastructure; D008962:Models, Theoretical; D055620:Optical Phenomena; D045663:Quantum Dots
D005456:Fluorescent Dyes