Photon-separation to enhance the spatial resolution of pulsed STED microscopy

Tortarolo, Giorgio; Sun, Yuansheng; Teng, Kai Wen; Ishitsuka, Yuji; Lanzanò, Luca; Selvin, Paul R; Barbieri, Beniamino; Diaspro, Alberto; Vicidomini, Giuseppe

doi:10.1039/c8nr07485b

Cited by 41 publications

(32 citation statements)

References 21 publications

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“…We conducted additional experiments, investigating further acquisition parameters such as the STED delay, pixel size and comparing the performance of different dyes to choose the optimal label (see Supplementary Material Section 7). STED microscopy critically depends on photophysics that can also be exploited to increase the resolution (lifetime 21 , photostability 23 , spectra 24 , etc.). For pulsed STED experiments, the best resolution is reached when the depletion pulses immediately follow the excitation pulse.…”

Section: Confocal/stedmentioning

confidence: 99%

Parameter-free image resolution estimation based on decorrelation analysis

2019

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Super-resolution microscopy opened diverse novel research directions by overcoming the classical resolution limit. Revealing structures beyond the diffraction limit was made possible by exploiting the fluorescent emission of individual fluorophores. Involving sample properties to apply these techniques entails a redefinition of the resolution parameter. Here, we propose a new method for assessing the resolution of individual super-resolved images based on image partial phase auto-correlation. The novel algorithm is model-free and does not require any user-defined parameters. We demonstrate its performance on a wide variety of imaging modalities, including diffraction-limited techniques. Finally, we show how our method can be used to optimize image acquisition and post-processing in superresolution microscopy.

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Section: Confocal/stedmentioning

confidence: 99%

Parameter-free image resolution estimation based on decorrelation analysis

2019

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“…The SPLIT approach has already been applied to enhance resolution in single-photon CW-STED microscopy 24,25 and fluctuation correlation spectroscopy 26 and, more recently, to pSTED microscopy. 27 Herein, we explore via the SPLIT method the spatially controlled gradients in fluorescence lifetime generated by the doughnut-shaped STED beam in a 2PE CW-STED microscope. In particular, to enhance SNR and SBR, the SPLIT approach implements an intelligent algorithm to simultaneously remove the STED background signal as well as the early photons originating from molecules at the periphery of the effective fluorescent volume, thus not effectively depleted/quenched.…”

Section: Introductionmentioning

confidence: 99%

Efficient two-photon excitation stimulated emission depletion nanoscope exploiting spatiotemporal information

et al. 2019

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Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking superresolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.

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“…Relying on a cell line stably expressing the mitochondrial protein COX8A fused to the SNAP‐tag and using STED nanoscopy, cristae of mitochondria in cultivated human cells with cell permanent dye SNAP‐Cell SiR are resolved with 70 nm resolution as shown in Figure 1e (Stephan et al, 2019). The pulsed STED microscopy with the separation by lifetime tuning (SPLIT) technique (pSTED‐SPLIT) is able to reveal the localization of the TOM20 proteins in the membrane of the ATTO 647N labeled mitochondria with higher spatial resolution (with respect to confocal results) (Tortarolo et al, 2019). Selected peroxisomal and mitochondrial membrane proteins—immunostained for PEX5, PEX14, PEX11, and TOM20, are imaged by using multicolor STED nanoscopy with 60 nm resolution (Galiani et al, 2016).…”

Section: Xy Dimensionsmentioning

confidence: 99%

Stimulated emission depletion microscopy for biological imaging in four dimensions: A review

2021

Microscopy Res & Technique

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Stimulated emission depletion (STED) microscopy allows high lateral and axial resolution, long term imaging in living cells. Here we review recent technical advances in STED microscopy, with emphasis on resolution and measurement range of XYZt four dimensions. Different STED technical advances and novel STED probes are discussed with their respective application in biological subcellular imaging. This review may serve as a practical guide for choosing a suitable approach to the advanced STED super‐resolution imaging.

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Photon-separation to enhance the spatial resolution of pulsed STED microscopy

Abstract: We introduce the pSTED-SPLIT microscopy technique, which explores the fluorescence dynamics in the phasor space to achieve sub-diffraction resolution images, with no need of high illumination intensity.

Cited by 41 publications

References 21 publications

Parameter-free image resolution estimation based on decorrelation analysis

Parameter-free image resolution estimation based on decorrelation analysis

Efficient two-photon excitation stimulated emission depletion nanoscope exploiting spatiotemporal information

Stimulated emission depletion microscopy for biological imaging in four dimensions: A review

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