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

Dertinger, Thomas; Colyer, Ryan A.; Iyer, Gopal; Weiss, Shimon; Enderlein, Jörg

doi:10.1073/pnas.0907866106

Cited by 1,017 publications

(1,233 citation statements)

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“…our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.In recent years the development of super-resolution techniques has had a profound impact on biology and other fields in which subdiffraction-limited resolution of fluorescently labeled samples is desired [1][2][3][4][5][6][7][8][9][10][11][12][13] . Prominent examples are stimulated emission depletion (STED) microscopy 1,4 , photoactivated localization microscopy (PALM) 3,5,6 and stochastic optical reconstruction microscopy (STORM) 2,7 .…”

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confidence: 99%

“…Whereas STED is based on a deterministic switching in a nanometric interference pattern, STORM and PALM are based on wide-field illumination and stochastic switching on the level of single isolated molecules, which are then localized. Other approaches, such as super-resolution optical fluctuation imaging (SOFI) 8 , reversible saturable optical fluorescence transitions (RESOLFT) microscopy 9,11 and saturated structured illumination microscopy (SSIM) 12,13 , are also based on stochastic or deterministic switching between two states and provide spatial resolution enhancement. Here we present an alternative approach that distinguishes adjacent molecules or nanoareas in the sample (arranged, for example, in a grid of 50 nm × 50 nm rectangular areas) by different average orientations of fluorescent dyes attached to rigid sample structures within these nanoareas.…”

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confidence: 99%

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Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

et al. 2014

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When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm) 2 image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, sPod). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, exPAn). this shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.In recent years the development of super-resolution techniques has had a profound impact on biology and other fields in which subdiffraction-limited resolution of fluorescently labeled samples is desired [1][2][3][4][5][6][7][8][9][10][11][12][13] . Prominent examples are stimulated emission depletion (STED) microscopy 1,4 , photoactivated localization microscopy (PALM) 3,5,6 and stochastic optical reconstruction microscopy (STORM) 2,7 . Generally, these techniques are based on reversible switching between two states. Whereas STED is based on a deterministic switching in a nanometric interference pattern, STORM and PALM are based on wide-field illumination and stochastic switching on the level of single isolated molecules, which are then localized. Other approaches, such as super-resolution optical fluctuation imaging (SOFI) 8 , reversible saturable optical fluorescence transitions (RESOLFT) microscopy 9,11 and saturated structured illumination microscopy (SSIM) 12,13 , are also based on stochastic or deterministic switching between two states and provide spatial resolution enhancement. Here we present an alternative approach that distinguishes adjacent molecules or nanoareas in the sample (arranged, for example, in a grid of 50 nm × 50 nm rectangular areas) by different average orientations of fluorescent dyes attached to rigid sample structures within these nanoareas. This is done by rotating the polarization of a wide-field excitation beam and detecting the periodic signals emitted with different phases from different nanoareas using wide-field camera detection (SPoD). We also show that the range of polarization angles that results in effective excitation of differently oriented molecules can be substantially narrowed by rotating a second wide-field de-exciting stimulated emission beam of a polarization perpendicular to the excitation beam polarization (ExPAN), resulting in better spatial resolution ...

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Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

et al. 2014

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“…The applicability of the photoswitch is, however, not restricted to PALM, but can also be used for reversible saturable optical fluorescence transitions (RESOLFT) microscopy [37] and super-resolution optical fluctuation imaging (SOFI). [38] The synthesis of these powerful photoswitches is straightforward, and a variety of substitutions by C À C coupling strategies is currently under way to specifically adapt their properties to the systems to be visualized. With suitable substitutions or embedding the chromophore into appropriate nanoparticles, we also envisage the possibility of expanding the applicability of the probe to imaging in polar media.…”

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Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

et al. 2016

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The in situ imaging of soft matter is of paramount importance for a detailed understanding of functionality on the nanoscopic scale. Although super-resolution fluorescence microscopy methods with their unprecedented imaging capabilities have revolutionized research in the life sciences, this potential has been far less exploited in materials science. One of the main obstacles for a more universal application of superresolved fluorescence microscopy methods is the limitation of readily available suitable dyes to overcome the diffraction limit. Here, we report a novel diarylethene-based photoswitch with a highly fluorescent closed and a nonfluorescent open form. Its photophysical properties, switching behavior, and high photostability make the dye an ideal candidate for photoactivation localization microscopy (PALM). It is capable of resolving apolar structures with an accuracy far beyond the diffraction limit of optical light in cylindrical micelles formed by amphiphilic block copolymers.The nanoscopic structure of soft-matter materials determines their properties. [1] Therefore, methods to directly visualize structures in the nanometer range are of paramount importance for the ongoing evolution of novel materials with specialized and adaptive properties for sophisticated applications. Scanning probe microscopy techniques give access to the nanometer range and determine surface properties such as topology and softness, [2,3] while modern electron microscopy methods, such as scanning electron microscopy (SEM) [4,5] and transmission electron microscopy (TEM), [6][7][8] can yield structural information even in the subnanometer range when there is sufficient electron density contrast. Despite the success of these methods, they are technically demanding and time-consuming. Furthermore, many softmatter samples possess poor electron contrast, and require non-invasive in situ imaging below the surface as well as the possibility to directly study dynamics. In recent years, superresolved fluorescence microscopy has revolutionized optical imaging, [9][10][11][12][13][14] by utilizing the photophysical or photochemical switching of fluorescent dyes in a sophisticated manner in combination with modern optics. So far, the life sciences have benefited, in particular, from the new possibilities of resolving structures well beyond the diffraction limit of light. Only a few examples of the application of super-resolution microscopy to materials science have been reported, [15][16][17][18] since concepts that require, for example, the addition of (polar) switching buffers often fail for these systems. Therefore, the main bottleneck for more universal applications of super-resolution imaging are switchable dyes with suitable (photo-)physical and chemical properties, such as high photostability, adjustable switching rates, minimum interaction with the environment to be probed, and simple design, with the possibility of multiple and straightforward derivatization for the specific labeling of structures or compartments. [19] ...

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“…3). [90,92,94] SIM SIM Patterned excitation allows information on fine details in the sample to be modulated onto the emitted pattern. Upon detection, the pattern is demodulated and the fine details are recovered.…”

Section: Stedmentioning

confidence: 99%

“…Methods based on the statistical analysis of fluorophore blinking have also proved very successful for molecular localisation in high molecular density images with improved temporal resolution [89][90][91][92]. These methods are described below as they make use of different spectroscopic properties.…”

Section: Temporal Resolution and Live-cell Stochastic Lmmentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

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Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

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

Cited by 1,017 publications

References 32 publications

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Nanoscopic Visualization of Soft Matter Using Fluorescent Diarylethene Photoswitches

Fluorescence nanoscopy. Methods and applications

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