Nanoscale resolution in GFP-based microscopy

Willig, Katrin I.; Kellner, Robert R.; Medda, Rebecca; Hein, Birka; Jakobs, Stefan; Hell, Stefan W.

doi:10.1038/nmeth922

Cited by 332 publications

(205 citation statements)

References 16 publications

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“…8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms. 9,10 STED nanoscopy uses stimulated emission to turn off the spontaneous fluorescence emission of dye molecules, typically overlapping a focused excitation beam with a "doughnut" shaped beam that deexcites emitters to the ground state everywhere except for the area within the centre of the doughnut, thus providing theoretically diffraction-unlimited resolution in the transverse plane by reducing the fullwidth half-maximum (FWHM) of the point spread function. By increasing the power of the depletion beam the emission region can be can be drastically reduced -theoretically allowing for sub-nanometre resolution -with resolutions of less than 10 nm being demonstrated.…”

Section: Plasmonic Nanoprobes For Stimulated Emission Depletion Nanosmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm that provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ~2x10 3 reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers and we also discuss their current limitations. Key-words: STED nanoscopy, nanorods, super-resolution, plasmonic nanoparticles, bio-imagingThe diffraction limit has ceased to be a practical limit to resolution in far-field microscopy, following the demonstration of STED, 1,2,3 RESOLFT 4 and localisation microscopies 5,6,7 and the subsequent development of a plethora of super-resolved nanoscopy techniques. 8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms. 9,10 STED nanoscopy uses stimulated emission to turn off the spontaneous fluorescence emission of dye molecules, typically overlapping a focused excitation beam with a "doughnut" shaped beam that deexcites emitters to the ground state everywhere except for the area within the centre of the doughnut, thus providing theoretically diffraction-unlimited resolution in the transverse plane by reducing the fullwidth half-maximum (FWHM) of the point spread function. By increasing the power of the depletion beam the emission region can be can be drastically reduced -theoretically allowing for sub-nanometre resolution -with resolutions of less than 10 nm being demonstrated. 11 The scaling of resolution with the square root of the depletion beam power means that relatively high-power lasers are typically used for STED nanoscopy. In practice, however, the use of high-power irradiation can result in problems such as photobleaching of the fluorophores and phototoxicity, and so the achievable resolution is compromised by the need to limit the intensity of the depletion laser radiation. Furthermore, high power lasers can add cost and complexity to STED microscopes and so the requirement for high power depletion beams presents challenges for parallelizing STED measurements 12,13 in terms of the lasers required, thus, limiting the potential for faster super-resolved imaging.To some extent the issue of photobleaching can be addressed with the development of more robust fluorescent labels such as quantum dots 14 as ...

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Section: Plasmonic Nanoprobes For Stimulated Emission Depletion Nanosmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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“…These ultrafast techniques open up fascinating new perspectives to explore molecular and cellular dynamics. Another exciting challenge for functional studies on cells will be to combine AFM molecular recognition imaging with advanced optical techniques, particularly stimulated emission depletion microscopy which currently reaches resolutions of a few tens of nanometers on cells [43].…”

Section: Discussionmentioning

confidence: 99%

Recent progress in AFM molecular recognition studies

Dufrêne

Hinterdorfer

2007

Pflugers Arch - Eur J Physiol

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During the past decade, remarkable advances have been made in using atomic force microscopy (AFM) for measuring the forces and the dynamics of the interaction between individual ligands and receptors, providing fundamental insights into molecular recognition processes. In addition, affinity imaging using either adhesion force mapping or dynamic recognition force mapping has offered a means to localize specific binding sites on model and cellular surfaces. These single-molecule analyses provide novel insight into the structure-function relationships of molecular recognition systems. In this review, we describe the principles of molecular recognition studies using AFM and provide a flavor of recent progress made in the field.

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“…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.…”

Section: Imaging Smaller: Pushing Resolution To the Limitmentioning

confidence: 99%

“…In the axial direction the resolution of a confocal microscope worsens due to the limited aperture angle which causes the extension of the focal spot in the optical axis to be about threeto fourfold larger than in the lateral direction [1]. In STED microscopy [66,75], in addition to the excitation beam a second doughnut-shaped beam is coupled into the illumination pathway. STED uses saturated stimulated depletion to transfer all excited fluorescent molecules back to the ground state, with the exception of the small volume present at the central focal point (i.e., the 'hole' of the doughnut), its size being nonlinearly related to the depletion intensity.…”

Section: Imaging Smaller: Pushing Resolution To the Limitmentioning

confidence: 99%