Time-gated stimulated emission depletion nanoscopy

Wang, Yifan; Kuang, Cuifang; Gu, Zhaotai; Xu, Yingke; Li, Shuai; Hao, Xiang; Xu, Liang

doi:10.1117/1.oe.52.9.093107

Cited by 26 publications

(12 citation statements)

References 18 publications

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“…Confocal scanning microscopy, one of the most widely used modern optical microscopy techniques, has enabled the enhancement of resolution by a factor of

\sqrt{2}

(Wilson, ; Gu, ). In the last 20 years, besides the method of confocal microscopy, several well‐known methods have been developed like stimulated emission depletion (STED) microscopy (Hell & Wichmann, ; Wang et al ., ), stochastic optical reconstruction microscopy (Rust et al ., ), photoactivated localization microscopy (PALM) (Betzig et al ., ) and structured illumination microscopy (Gustafsson, ). In STED microscopy, the sample is illuminated with a pump beam, and almost simultaneously the STED beam suppresses the fluorescence process at the periphery around the centre through simulated emission.…”

Section: Introductionmentioning

confidence: 99%

Image scanning fluorescence emission difference microscopy based on a detector array

Liu

et al. 2017

Journal of Microscopy

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We propose a novel imaging method that enables the enhancement of three-dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.

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“…Confocal scanning microscopy, one of the most widely used modern optical microscopy techniques, has enabled the enhancement of resolution by a factor of

\sqrt{2}

Section: Introductionmentioning

confidence: 99%

Image scanning fluorescence emission difference microscopy based on a detector array

Liu

et al. 2017

Journal of Microscopy

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“…reducing the photodamage and the phototoxicity. 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].…”

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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“…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 . However, this approach suppresses the valuable signal from the focal center, occurring within the time the detector is gated off, which makes it difficult to improve the effective resolution in the all‐pulsed 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

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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.

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Time-gated stimulated emission depletion nanoscopy

Cited by 26 publications

References 18 publications

Image scanning fluorescence emission difference microscopy based on a detector array

Image scanning fluorescence emission difference microscopy based on a detector array

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

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

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