SOFI: Turning fluorescence fluctuations into improved optical resolution
(published March 25, 2010) – Fluorescence microscopy permits the three-dimensional imaging of living cells, tissues and even small organisms. However, features smaller than approximately half the emission wavelength (~200 – 300 nm) cannot be resolved in conventional far-field microscopy due to the optical diffraction limit. Other techniques, such as electron microscopy and scanning probe microscopy, achieve molecular-level resolution, but are not suitable for imaging features within live cells. During the last decade, the optical diffraction limit has been overcome with the introduction of several new concepts, pioneered by stimulated emission depletion microscopy, ground state depletion microscopy, (saturated) structured illumination microscopy, and image interference microscopy. Stochastic techniques using photoswitchable probes have also been developed such as photo-activated localization microscopy, stochastic optical reconstruction microscopy, and variants thereof. All these superresolution methods are capable of enhancing the resolution in 3D, but often at the expense of major technical demands or modifications to the microscope.
Figure 1: Left: Conventional wide-field image of quantum dot labelled microtubules of a 3T3-fibroblast. This image was acquired using a lamp as an excitation source and detecting the infrared fluorescence (~800 nm) using a sensitive CCD-camera. Right: SOFI image, having a two-fold improved resolution compared to the original image. On top of the resolution enhancement along all three dimensions, the SOFI algorithm annihilates any background signal and therefore leads to an enhanced contrast throughout the image.
A joint collaboration of scientists form the University of California in Los Angeles and the Georg-August University in Göttingen has developed a new 3D superresolution method which not only overcomes the diffraction limit, but also generates virtually background-free, contrast-enhanced images with a few seconds of acquisition time. It is based on the analysis of temporal fluorescence fluctuations of emitters (such as the blinking of quantum dots or the triplet state blinking of conventional fluorescent dyes), which they termed superresolution optical fluctuation imaging or SOFI. The core idea of SOFI is that the blinking of different emitters in a sample such as a labeled cell is statistically independent from each other, enabling to spatially disentangle the different fluorescence contributions from different emitters in a given image.. The only hardware prerequisite of this method is the ability to record with high sensitivity and speed consecutive fluorescence images of a sample. Resolution improvement is achieved by evaluating the temporal fluorescence fluctuations in the recorded movie using a clever software algorithm. This makes SOFI exceptionally simple from a hardware point of view, because virtually any wide-field microscope equipped with a fast and sensitive camera can be converted into a superresolution microscope. In the paper, the authors apply the method to the imaging of a cell’s cytoskeleton and demonstrate its power in resolution improvement and background suppression.
T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein
“Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)” Proc. Nat. Acad. Sci. USA (2009) 106(52):22287-22292.
Source: Prof. Dr. Joerg Enderlein, Georg August University, Göttingen, Germany Contact:
Prof. Dr. Joerg Enderlein
Georg August University
Department of Physics
III. Institute of Physics
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