Super-resolution video microscopy of live cells by structured illumination

Kner, Peter; Chhun, Bryant B.; Griffis, Eric R.; Winoto, Lukman; Gustafsson, Mats

doi:10.1038/nmeth.1324

Cited by 694 publications

(542 citation statements)

References 23 publications

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“…The key element common to these super-resolution techniques for breaking the diffraction limit is the use of the photoswitching property of fluorophores, which is realized by using various optical phenomena such as stimulated emission, cis-trans isomerization, triplet pumping [14] and saturated excitation (SAX) [15][16][17]. Structured illumination microscopy (SIM), while not breaking the confocal microscopy diffraction limit, is also well known as a high-resolution imaging technique which doubles the spatial resolution by overcoming the diffraction limit in classical wide-field fluorescence microscopy [18,19].…”

Section: Introductionmentioning

confidence: 99%

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

et al. 2013

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We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.

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Section: Introductionmentioning

confidence: 99%

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

et al. 2013

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“…STED has achieved video rate (13) but the method is quite demanding in terms of labeling procedures and choice of dyes and requires tedious alignment procedures. Recently, superresolution microscopy at 11 Hz has been demonstrated by using SIM, achieving a 2-fold increased lateral resolution (14). All super-resolution methods are capable of enhancing resolution in 3D, but often at the expense of major technical demands or modifications to the microscope (15)(16)(17).…”

mentioning

confidence: 99%

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

Dertinger

Colyer²,

Iyer³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

1,018

1,167

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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.cumulants ͉ fluorescence ͉ quantum dots ͉ superresolution microscopy ͉ intermittency

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“…Another approach for investigating dynamic processes with subdiffraction resolution is based on stochastically switching and tracking single molecules [22], which was demonstrated in 2007 [23] and 2008 [24]. So-called structured illumination microscopy, with diffraction-limited resolution of 100 nm, was recently used for dynamic imaging with 11 frames per second in combination with TIRF illumination [25].…”

Section: Biophotonicsmentioning

confidence: 99%

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

Lauterbach

Keller

Schönle

et al. 2010

Journal of Biophotonics

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We compare the performance of video‐rate Stimulated Emission Depletion (STED) and confocal microscopy in imaging the interior of living neurons. A lateral resolution of 65 nm is observed in STED movies of 28 frames per second, which is 4‐fold higher in spatial resolution than in their confocal counterparts. STED microscopy, but not confocal microscopy, allows discrimination of single features at high spatial densities. Specific patterns of movement within the confined space of the axon are revealed in STED microscopy, while confocal imaging is limited to reporting gross motion. Further progress is to be expected, as we demonstrate that the use of continuous wave (CW) beams for excitation and STED is viable for video‐rate STED recording of living neurons. Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Super-resolution video microscopy of live cells by structured illumination

Cited by 694 publications

References 23 publications

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

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

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

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