Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell

Abstract

We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed-and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Timelapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.fluorescence ͉ GFP ͉ microscopy ͉ resolution

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

“…Indeed, a comparison of our microtubule images with images obtained by other super-resolution techniques 2,21 demonstrates that this resolution variation is similar to that observed with STED or PALM, which are also subject to local sample variations in fluorescence lifetimes or label densities. Nevertheless, it is always advantageous to choose an image pixel size that is similar to or somewhat larger than the worst local resolution in the entire image, thereby ensuring a homogeneous resolution throughout the image.…”

“…1), the experimental examples presented here (Figs. 2-4) show that the observed high-resolution images are at least as homogeneous as those of other high-resolution techniques 2,21,26 . In the future, the approach could be extended to three-dimensional demodulation including the z axis of the molecular orientation.…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…Finally, the third aspect of imaging that needs to be addressed is resolution, where we find great efforts directed towards super-resolution techniques, including stimulated emission depletion (STED) [63][64][65][66], structured illumination microscopy (SIM) [67,68], photoactivated localization microscopy (PALM) [69,70], and stochastic optical reconstruction microscopy (STORM) [71][72][73][74]. These approaches offer sub-diffraction-limited resolution and have opened new ways of exploring the submicron world within the living cell.…”

Fluorescence nanoscopy. Methods and applications

“…STED imaging can be performed on fusion constructs of fluorescent proteins that also span most of the visible spectrum: green (GFP [112]), yellow (YFP [113] and citrine [114]) and far-red emitting proteins (TagRFP657 [115] and the tetrametric E2-Crimson [116]). Several reversible photoswitchable proteins have also been demonstrated suitable for RESOLFT imaging, exploiting their reversible switching behaviour to deplete the periphery of the focal spot (Dronpa and its counterpart Padron derivative [15,22], rsEGFP2 [23] and Dreiklang [117]).…”

“…The endoplasmic reticulum (ER) was one of the first organelles targeted by STED imaging: The fluorescent protein citrine concentrated on the ER permitting volumetric imaging of this organelle [114]. The Zhuang lab has also very recently demonstrated super-resolution STORM imaging with membrane probes, such as carbocyanine dyes with alkyl chains (DiI, DiD, DiR), cationic dyes (MitoTracker) and BODIPY conjugated probes (Er/LysoTracker) specific to the plasma membrane, mitochondria and ER or lysosomes, respectively, capturing details of morphological dynamics of mitochondrial fusion and fission and ER remodelling [172].…”