Background suppression in fluorescence nanoscopy with stimulated emission double depletion

Gao, Peng; Prunsche, Benedikt; Zhou, Lu; Nienhaus, Karin; Nienhaus, G. Ulrich

doi:10.1038/nphoton.2016.279

Cited by 117 publications

(123 citation statements)

References 31 publications

Supporting

Mentioning

123

Contrasting

Order By: Relevance

“…Commercial, dye‐loaded polystyrene NPs (PS NPs) with different surface functionalization have found widespread application in the nanotechnology field, e. g., as biosensors or for self‐assembling nanostructures . Red‐emitting 40 nm PS NPs are excellent tools to precisely assess the performance of advanced imaging techniques such as stimulated emission double depletion (STEDD) nanoscopy ,. In this differential STED variant, a first, donut‐shaped STED pulse depletes the periphery of the excitation spot and is followed by a second, Gaussian STED pulse that specifically depletes the central region of the excitation spot, thus leaving only background.…”

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

“…In this differential STED variant, a first, donut‐shaped STED pulse depletes the periphery of the excitation spot and is followed by a second, Gaussian STED pulse that specifically depletes the central region of the excitation spot, thus leaving only background. For each pixel, this background is subtracted from the initial STED image to obtain background‐free super‐resolved images (Figure ) ,. Of note, STEDD also offers enhanced flexibility in the choice of depletion lasers as it permits the use of lower‐power, less red‐shifted depletion pulses.…”

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

“…used commercial QDs (Qdot705, Thermo Fisher, Waltham, MA) to show that background due to re‐excitation by the STED beam can be removed by taking a first raster‐scan image with excitation and STED beam illumination, and a second one with STED beam illumination only. Subsequent subtraction of the two images cancels the re‐excitation background, very much in the vein of the STEDD approach ,. However, STEDD nanoscopy is advantageous because it takes the pixel intensities contributing to the two images within nanoseconds and also removes background arising from incomplete depletion .…”

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

“…Subsequent subtraction of the two images cancels the re‐excitation background, very much in the vein of the STEDD approach ,. However, STEDD nanoscopy is advantageous because it takes the pixel intensities contributing to the two images within nanoseconds and also removes background arising from incomplete depletion . Yang et al .…”

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

See 3 more Smart Citations

Nanoparticle Probes for Super‐Resolution Fluorescence Microscopy

Lin

Nienhaus

2018

ChemNanoMat

Self Cite

View full text Add to dashboard Cite

The Abbe resolution limit of ∼200 nm of conventional light‐optical microscopy greatly restricts the observation of subcellular processes. This Abbe resolution barrier has been surpassed by super‐resolution fluorescence microscopy methods that use time as an additional dispersing dimension. Light irradiation can be employed to externally control the emission properties of the luminescent markers, or spontaneous fluctuations of the emission of individual emitters can be analyzed. Here we review the development and application of light‐emitting nanoscale particles for super‐resolution imaging and discuss their (photo)physical and (photo)chemical characteristics to offer guidance in the selection of the optimal nanoparticle marker for an intended super‐resolution imaging experiment.

show abstract

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

Section: Nanoparticles For Super‐resolution Fluorescence Microscopymentioning

confidence: 99%

See 2 more Smart Citations

Nanoparticle Probes for Super‐Resolution Fluorescence Microscopy

Lin

Nienhaus

2018

ChemNanoMat

Self Cite

View full text Add to dashboard Cite

show abstract

“…Recently, several methods have been proposed to reduce the required depletion power for STED super‐resolution imaging, such as a time‐gated approach, the synthesis of new luminescence materials with lower saturation intensity and the use of a wavelength closer to the emission maximum . The time‐gated approach is a well‐established method to improve the fluorescence on–off contrast in STED imaging, which can strongly suppress low spatial frequencies in continuous‐wave STED modality .…”

Section: Introductionmentioning

confidence: 99%

Elimination of Re‐excitation in Stimulated Emission Depletion Nanoscopy Based on Photon Extraction in a Phasor Plot

Chen

Wang

Yan

et al. 2020

Laser & Photonics Reviews

View full text Add to dashboard Cite

Stimulated emission depletion (STED) nanoscopy can achieve super‐resolution imaging of microstructures at the nanoscale by confining the fluorescence emission of a diffraction‐limited focusing spot. The depletion wavelength is typically at the red tail of the emission spectrum of the fluorophore to avoid re‐excitation; however, inefficient fluorescence inhibition on account of the small stimulated emission cross‐section results in a huge demand for the depletion of laser power. Here, an accurate photon extraction method is proposed to eliminate re‐excitation caused by blue shift of the depletion wavelength. After only extracting the photons unaffected by a donut‐shaped depletion laser in the phasor plot, both the resolution and signal‐to‐noise ratio of the STED images are significantly improved. As a result, the increased depletion efficiency at a blue shifted depletion wavelength reduces the power demand for achieving a certain resolution. This approach, with flexible wavelength selectivity owing to the independence of depletion power, made more fluorophores applicable for STED super‐resolution imaging, thereby offering more convenient and efficient service to researchers.

show abstract

Stimulated Emission Depletion Microscopy

Li¹,

Wang²,

Kuang³

et al. 2021

Encyclopedia of Life Sciences

View full text Add to dashboard Cite

As one of the mainstream methods of super‐resolution microscopy, stimulated emission depletion (STED) microscopy can effectively break through the diffraction limit of traditional far‐field optical microscopy and increase the resolution of the microscope to the level of nanometre. In the course of about 20 years, STED microscopy has continued to get evolved and has been widely used in many fields, such as life science and physical research, which effectively broadens the scope of our understanding of the microworld. We believe this review will provide a guide for broad readers in related technology. Key Concepts STED microscopy can achieve three‐dimensional super‐resolution imaging with a common resolution of 10–70 nm. Parallelisation illumination can achieve high‐speed imaging using multiple excitation foci to illuminate the specimens. Conventional STED imaging in deep tissue remains challenging mostly due to the fact that light gets scattered in tissues. Under ultrahigh depletion radiation, background signals are generated by secondary excitation of the depletion illumination which has so far prohibited STED microscopy from reaching its theoretically molecular resolution. In practice, the attainment of a higher resolution is notably retarded by the existence of photobleaching effect, a kind of photochemical reaction that permanently inhibits fluorophore molecules to fluoresce.

show abstract

Background suppression in fluorescence nanoscopy with stimulated emission double depletion

Cited by 117 publications

References 31 publications

Nanoparticle Probes for Super‐Resolution Fluorescence Microscopy

Nanoparticle Probes for Super‐Resolution Fluorescence Microscopy

Elimination of Re‐excitation in Stimulated Emission Depletion Nanoscopy Based on Photon Extraction in a Phasor Plot

Stimulated Emission Depletion Microscopy

Contact Info

Product

Resources

About