STED with wavelengths closer to the emission maximum

Vicidomini, Giuseppe; Moneron, Gael; Eggeling, Christian; Rittweger, Eva; Hell, Stefan W.

doi:10.1364/oe.20.005225

Cited by 97 publications

(114 citation statements)

References 37 publications

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“…Another possibility would be to reduce the depletion intensity required to deplete the dye by working at shorter wavelengths. 37,38 Specifically, designing anisotropic NPs with a LSPR centered at ~750 nm, where the cross-section for stimulated emission depletion of STAR635P is approximately 3 times higher than at 780nm, could reduce the intensity required to achieve a given STED resolution by the same factor of ~3, thus, further reducing the heat generated. Engineering smaller plasmonic particles could also reduce the amount of heat generated, however, the field-enhancements obtained with such particles would be lower.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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“…Since the anti-Stokes fluorescence background is triggered solely by the STED beam, it has been recently demonstrated that a lock-in (synchronous) detection system can effectively subtract this background [9,10]. Per contra, when the lock-in detection is applied for imaging, it doubles the recording time.…”

Section: Biophotonicsmentioning

confidence: 99%

“…Our implementation is based on a TCSPC card, which is commonly used in gCW-STED microscopy [8,15], but an alternative implementation can use two hardware time-gates. Similarly to the lock-in detection methods the success of the proposed method depends on the reliability of the background estimation, which increases with the increase of the pixel dwell-time [9]. Thereby, as with the the lock-in methods, this method also needs the careful choice of the pixel dwell-time.…”

Section: Biophotonicsmentioning

confidence: 99%

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Hernández

Peres

Zanacchi

et al. 2014

Journal of Biophotonics

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Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…It has been observed before that loss of fluorescence may be accelerated by the presence of a depletion source [12,26]. This may eventually affect the SNR that can be achieved in the images.…”

Section: Images In Cell Samplesmentioning

confidence: 96%

“…In practice, high depletion laser power can have a negative effect on the sample and on the quality of the final image. This is because high depletion power can result in increased photobleaching, blinking, re-excitation or two-photon effects [9][10][11][12][13][14]. All these interactions of the excitation and depletion beams with the sample are specific to the fluorescent molecule used and the sample itself, and they may affect the quality of the image.…”

Section: Introductionmentioning

confidence: 99%

STED imaging performance estimation by means of Fourier transform analysis

Merino¹,

Mallabiabarrena²,

Andilla³

et al. 2017

Biomed. Opt. Express

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Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze the performance of a STED system. The spatial frequency distribution of pairs of confocal and STED images are compared to obtain an objective parameter, the Azimuth Averaged Spectral Content Spread (AASCS), that is related to the performance of the system in which the sample is also considered. The method has been first tested on samples of beads, and then applied to cell samples labeled with multiple fluorescent dyes. The results show that a single parameter, the AASCS, can be used to determine the optimal settings for STED image acquisition in an objective way, only by using the information provided by the images from the sample themselves. The AASCS also helps minimize the depletion power, for better preservation of the samples.

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STED with wavelengths closer to the emission maximum

Cited by 97 publications

References 37 publications

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

STED imaging performance estimation by means of Fourier transform analysis

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