Ultrafast photon counting applied to resonant scanning STED microscopy

Wu, Xundong; Toro, Ligia; Stefani, Enrico; Wu, Yong

doi:10.1111/jmi.12183

Cited by 21 publications

(16 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It was testified that with ࣙ6 GW cm -2 depletion irradiance, the fluorophore survival time was extended by ß80%, as well as an 8-fold wider field of view (FOV). What's more, an ultrafast photon counting system was developed that delivered a 450 MHz pixel clock (2.2 ns pixel dwell time in each scan), obtaining a large FOV (Wu et al, 2015b). They also advanced the time-gated CW STED technique to provide superb signal to noise ratio (SNR) and finer image quality.…”

Section: Fast Scanningmentioning

confidence: 99%

Recent research on stimulated emission depletion microscopy for reducing photobleaching

Liu

Wang

et al. 2018

Journal of Microscopy

View full text Add to dashboard Cite

Stimulated emission depletion (STED) microscopy is a useful tool in investigation for super-resolution realm. By silencing the peripheral fluorophores of the excited spot, leaving only the very centre zone vigorous for fluorescence, the effective point spread function (PSF) could be immensely squeezed and subcellular structures, such as organelles, become discernable. Nevertheless, because of the low cross-section of stimulated emission and the short fluorescence lifetime, the depletion power density has to be extremely higher than the excitation power density and molecules are exposed in high risk of photobleaching. The existence of photobleaching greatly limits the research of STED in achieving higher resolution and more delicate imaging quality, as well as long-term and dynamic observation. Since the first experimental implementation of STED microscopy, researchers have lift out variety of methods and techniques to alleviate the problem. This paper would present some researches via conventional methods which have been explored and utilised relatively thoroughly, such as fast scanning, time-gating, two-photon excitation (TPE), triplet relaxation (T-Rex) and background suppression. Alternatively, several up-to-date techniques, especially adaptive illumination, would also be unveiled for discussion in this paper. The contrast and discussion of these modalities would play an important role in ameliorating the research of STED microscopy.

show abstract

Section: Fast Scanningmentioning

confidence: 99%

Recent research on stimulated emission depletion microscopy for reducing photobleaching

Liu

Wang

et al. 2018

Journal of Microscopy

View full text Add to dashboard Cite

show abstract

“…In our STED microscope we use a home-built ultrafast photo-counting acquisition system with up to 450 MHz pixel clock rate [21]. In this home-built system, time-gated detection is implemented by hardware.…”

Section: Microscope Assemblymentioning

confidence: 99%

“…Since we adopt resonant scanning, the first step of image processing is to correct for the distortion caused by the sinusoidal movement of the resonant mirror, which has been discussed in our previous publications [12,21]. Here we focus on background/noise reduction and performing statistics on clusters for their localization and colocalization.…”

Section: Image Processing and Analysismentioning

confidence: 99%

“…The green channel implements time-gated detection to boost resolution. To reach maximal signal-to-noise ratio, we adopted photon counting for acquisition, and we built a photon-counting system with ultrafast pixel clock rate (or count rate) to keep up with the fast scanning speed [21]. In this paper, we share our experience in building a resonant-scanning dual-channel STED microscope and its applications to imaging biological samples.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

Toro

et al. 2015

Methods

Self Cite

View full text Add to dashboard Cite

STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.

show abstract

“…For these reasons, a trade-off between photobleaching and effective resolution must be reached when choosing the pulse-width of the STED beam. Early STED implementations obtained such a compromise with 100-200 ps pulse-width (Gould, Myers, & Bewersdorf, 2011;Vicidomini, Coto Hern andez, Diaspro, Galiani, & Eggeling, 2015a;Wu, Toro, Stefani, & Wu, 2015a).…”

mentioning

confidence: 99%

Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

Castello

Tortarolo

Hernández

et al. 2016

Microsc. Res. Tech.

View full text Add to dashboard Cite

The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal-to-noise ratio. Typically, an increase of the STED beam's power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beam's power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P-STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beam's power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time-gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost.

show abstract

Ultrafast photon counting applied to resonant scanning STED microscopy

Cited by 21 publications

References 20 publications

Recent research on stimulated emission depletion microscopy for reducing photobleaching

Recent research on stimulated emission depletion microscopy for reducing photobleaching

Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

Contact Info

Product

Resources

About