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

Lauterbach, Marcel A.; Keller, Jan; Schönle, Andreas; Kamin, Dirk; Westphal, Volker; Rizzoli, Silvio O.; Hell, Stefan W.

doi:10.1002/jbio.201000038

Cited by 42 publications

(25 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

mentioning

confidence: 99%

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.

304

253

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 99%

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.

304

253

View full text Add to dashboard Cite

show abstract

“…Since the depletion beam has more impact on the resolution, we only did aberration correction with the SLM on the depletion beam. Previous published papers [21–23] used the total image brightness (sum of pixel values) as an image quality metrics for finding correction phases in confocal and two-photon microscopy systems. In STED system, Gould et al used image brightness and sharpness as the quality metrics of PSFs [12].…”

Section: Aberration Correctionmentioning

confidence: 99%

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

et al. 2017

View full text Add to dashboard Cite

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens’ optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples.

show abstract

“…Increasing scanning speed requires excitation and depletion powers to be increased accordingly, and therefore, photodamage and photobleaching become a major concern. Video-rate (28 Hz) STED imaging at 65 nm lateral resolution has been demonstrated on living cells, although the field of view was relatively small (4.5 μm 2 ), and it was confined to two dimensions possibly due to out-of-focus photodamage, which precluded from whole-cell imaging [26,27]. In an impressive demonstration, in vivo STED of the brain of a living mouse has been recently demonstrated [28].…”

Section: Temporal Resolution and Live-cell Sted/resolft Imagingmentioning

confidence: 99%

“…Fluorescence nanoscopy makes possible the investigation of the nanoscale dynamic organisation of proteins within subsynaptic domains: The fate of synaptic vesicles after exocytosis and fusion with the membrane has been studied by STED microscopy in fixed [199,200] and live neurons using video-rate (20)(21)(22)(23)(24)(25)(26)(27)(28) [27,201] and time-lapse STED microscopy [202,203]. AMPA receptors trafficking in neuronal cells have been investigated by single particle tracking PALM [204].…”

Section: Neurobiologymentioning

confidence: 99%

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

View full text Add to dashboard Cite

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.

show abstract

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

Cited by 42 publications

References 33 publications

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

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

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

Fluorescence nanoscopy. Methods and applications

Contact Info

Product

Resources

About