Effects of aberrations on effective point spread function in STED microscopy

Li, Yanghui; Zhou, Hui; Liu, Xiaoyu; Li, Yuxue; Wang, Le

doi:10.1364/ao.57.004164

Cited by 18 publications

(16 citation statements)

References 15 publications

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“…In accordance with previous work on the effect of aberrations on STED depletion patterns, 17 , 23 we found that spherical aberrations were detrimental to z-STED but had very limited effects in 2D-STED ( Figure 6 c). Surprisingly, spherical aberrations slightly decreased the average number of molecules in the observation volume in 2D-STED, which can be attributed to an elongation of the depletion pattern (insets in Figure 6 a,b), leading to a better overlap between excitation and depletion foci.…”

Section: Resultssupporting

confidence: 93%

Background Reduction in STED-FCS Using a Bivortex Phase Mask

et al. 2020

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Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics in living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes on the nanoscale in living cells. In two-dimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volume, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern created by a bivortex phase mask reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS-based investigations of 3D diffusion on the subdiffraction scale.

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Section: Resultssupporting

confidence: 93%

Background Reduction in STED-FCS Using a Bivortex Phase Mask

et al. 2020

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“…In accordance with previous work on the effect of aberrations on STED depletion patterns [11], [21], we found that spherical aberrations were detrimental to z-STED but had very limited effects in 2D STED (Figure 7(c)). Suprisingly, spherical aberrations slightly decreased the average number of molecules in the observation volume in 2D STED, which can be attributed to an elongation of the depletion pattern (insets in Figure 7(a)-(b)), leading to a better overlap between excitation and depletion foci.…”

Section: Figure 7: Effect Of Common Aberrations On the Depletion Pattsupporting

confidence: 93%

“…The employed depletion patterns show different sensitivity to optical aberrations [11], [20], [21]. To ensure optimal performance and thus fair comparison of the confinement modes, we first corrected sample-induced optical aberrations, as well as residual system-induced aberrations, using the sensorless adaptive optics approach described in [13].…”

Section: Adaptive Opticsmentioning

confidence: 99%

Background reduction in STED-FCS using coherent-hybrid STED

Barbotin

Urbančič

Galiani

et al. 2020

Preprint

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Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics of living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes at the nanoscale in living cells. In twodimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volumee, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS based investigations of 3D diffusion on the sub-diffraction scale.

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“…In principle, the z-STED depletion pattern should greatly simplify the analysis and interpretation of STED-FCS measurements of 3D diffusion. However, the main factor limiting the use of z-STED-FCS is the extreme sensitivity to optical aberrations [ 11 – 13 ] that can fill up the central intensity minimum or distort the focus, reducing brightness and resolution [ 14 ] and ultimately leading to an amount of noise precluding reliable STED-FCS measurements.…”

Section: Introductionmentioning

confidence: 99%

Adaptive optics allows STED-FCS measurements in the cytoplasm of living cells

Barbotin¹,

Galiani²,

Urbančič³

et al. 2019

Opt. Express

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Fluorescence correlation spectroscopy in combination with super-resolution stimulated emission depletion microscopy (STED-FCS) is a powerful tool to investigate molecular diffusion with sub-diffraction resolution. It has been of particular use for investigations of two dimensional systems like cell membranes, but has so far seen very limited applications to studies of three-dimensional diffusion. One reason for this is the extreme sensitivity of the axial (z) STED depletion pattern to optical aberrations. We present here an adaptive optics-based correction method that compensates for these aberrations and allows STED-FCS measurements in the cytoplasm of living cells. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article's title, journal citation, and DOI.

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Effects of aberrations on effective point spread function in STED microscopy

Cited by 18 publications

References 15 publications

Background Reduction in STED-FCS Using a Bivortex Phase Mask

Background Reduction in STED-FCS Using a Bivortex Phase Mask

Background reduction in STED-FCS using coherent-hybrid STED

Adaptive optics allows STED-FCS measurements in the cytoplasm of living cells

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