Super-resolution 3D microscopy of live whole cells using structured illumination

Shao, Lin; Kner, Peter; Rego, E. Hesper; Gustafsson, Mats

doi:10.1038/nmeth.1734

Cited by 477 publications

(372 citation statements)

References 18 publications

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“…Only the cristae with relatively large spacing appeared to be resolved in the live-cell STORM images (Fig. 4), as in previous live-cell images by structured illumination microscopy (46). The more tightly packed cristae with smaller spacing, observed in EM and fixed-cell STORM images (Fig.…”

Section: Discussionsupporting

confidence: 75%

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Shim

Xia

Zhong

et al. 2012

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

466

455

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Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.nanoscopy | diffraction limit | photoswitchable dye | stochastic optical reconstruction microscopy | photoactivation localization microscopy

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Section: Discussionsupporting

confidence: 75%

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Shim

Xia

Zhong

et al. 2012

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

466

455

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“…S-polarization in the excitation beams, required for achieving a high contrast ratio in the illumination pattern regardless of pattern orientations, was achieved by a linear polarizer corotating with the grating. More recently, faster setups have been published that use fast spatial light modulators (SLMs) for the pattern generation (9,11,18,19) and liquid crystal waveplates for fast polarization rotation (11,20). Live-cell imaging has been demonstrated in two dimensions using total internal reflection fluorescence (TIRF) (11) and, more recently, in three dimensions (9, 20), but both in only one color.…”

mentioning

confidence: 99%

“…More recently, faster setups have been published that use fast spatial light modulators (SLMs) for the pattern generation (9,11,18,19) and liquid crystal waveplates for fast polarization rotation (11,20). Live-cell imaging has been demonstrated in two dimensions using total internal reflection fluorescence (TIRF) (11) and, more recently, in three dimensions (9,20), but both in only one color. The limitation to one excitation wavelength stems mainly from the fact that the generation of the illumination beams used for structured illumination is based on diffraction, which is wavelengthdependent.…”

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confidence: 99%

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Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Fiolka

Shao

Rego

et al. 2012

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

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308

253

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Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it lightefficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.extended resolution | frequency mixing | multicolor | patterned excitation F luorescence microscopy allows noninvasive 3D imaging of the interior of living specimens with molecular specificity, and is therefore an invaluable resource to the biological sciences. Unfortunately, the resolving power of fluorescence microscopy is fundamentally limited by the diffraction of light.Lately, many techniques have been introduced to extend the resolution beyond the classic diffraction limit (1-8). Although the improvement of spatial resolution is impressive, the practical impact of these new techniques will largely depend on whether they can keep the two key advantages of fluorescence microscopy, namely, the capability of imaging living cells in three dimensions and multicolor labeling. Live-cell imaging over many time points has been demonstrated for some resolution-extending techniques (9-15) but, to our knowledge, not for multicolor 3D imaging of whole cells.Stimulated emission depletion (STED) microscopy increases the spatial resolution by suppressing the fluorescence emission on the rim of a focused laser spot, theoretically enabling unlimited resolution (16). STED microscopy has been demonstrated for live imaging at high frame rates (12, 15), but its point-scanning nature makes it unsuitable for fast volumetric imaging over large fields of view. Furthermore, to achieve high spatial resolution, very high power densities (38-540 MW/cm 2 ) (12) are necessary for the STED laser beam, which may cause increased photobleaching and phototoxicity, thus limiting the application of live-cell STED microscopy.In localization-based microscopy, only sparse subsets of photoswitchable fluorophores in a sample are imaged at a time, which allows the isolation and precise localizati...

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“…After replacing the optomechanics for a spatial light modulator, 3D SIM has been demonstrated on living cells with low light intensities of (≈5 W cm −2 ), requiring only about 5 to 25 s per complete cell [105]. Spatial resolution was twice that of conventional microscopy, and temporal resolution was only limited by the CCD read-out time.…”

Section: D Sim Temporal Resolution and Live-cell Imagingmentioning

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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Super-resolution 3D microscopy of live whole cells using structured illumination

Cited by 477 publications

References 18 publications

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination

Fluorescence nanoscopy. Methods and applications

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