easySLM‐STED: Stimulated emission depletion microscopy with aberration correction, extended field of view and multiple beam scanning

Görlitz, Frederik; Guldbrand, Stina; Runcorn, T. H.; Murray, R.; Jaso-Tamame, Angel Luis; Sinclair, Hugo G.; Martínez-Pérez, Enrique; Taylor, J.R.; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.

doi:10.1002/jbio.201800087

Cited by 26 publications

(24 citation statements)

References 22 publications

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“…When combined with iSIM imaging, an expansion factor of ∼4 results in an effective resolution of ∼140 / 4 = 35 nm laterally and ∼350 / 4 ≈ 90 nm axially after deconvolution. Compared with current state-of-the-art super-resolution microscopes with similar resolution performance, such as the HT-STORM 10 capable of imaging a 100×100 µm 2 FOV in ∼5-10 minutes or a similar FOV with easySLM-STED 32 in 60-80 minutes, the iSIM is capable of stitching together images of expanded samples to form an equivalent FOV within 2-5 seconds. This represents a 100-1000-fold improvement in imaging throughput.…”

Section: Resultsmentioning

confidence: 99%

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mahecic

Gambarotto

Douglass

et al. 2020

Preprint

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19Super-resolution microscopies, which allow features below the diffraction limit to be 20 resolved, have become an established tool in biological research. However, imaging 21 throughput remains a major bottleneck in using them for quantitative biology, which requires 22 large datasets to overcome the noise of the imaging itself and to capture the variability 23 inherent to biological processes. Here, we develop a multi-focal flat illumination for field 24 independent imaging (mfFIFI) module, and integrate it into an instant structured illumination 25 microscope (iSIM). Our instrument extends the field of view (FOV) to >100x100 µm 2 without 26 compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric 27 imaging at double the diffraction-limited resolution. We further extend the effective FOV by 28 stitching multiple adjacent images together to perform fast live-cell super-resolution imaging 29 of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure 30 expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human 31 cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at 32 an effective resolution of ~35 nm. We apply classification and particle averaging to these 33 large datasets, allowing us to map the 3D organization of post-translational modifications of 34 centriolar microtubules, revealing differences in their coverage and positioning. 35 (STED) 5 . Initial implementations of these methods were relatively slow, in part due to the 42 use of the time domain to separate single molecules (SMLM), the need to collect multiple 43 images to cover Fourier space (SIM), or the scanning of a reduced excitation volume 44 3 (STED). In both wide-field and patterned illumination, the imaging throughput -defined as 45 the area imaged per time -can be increased by parallelizing the acquisition, as long as the 46 necessary illumination can be maintained over a larger surface in the sample plane. 47Extending the array of patterned excitation has been used to increase the speed or FOV of 48 confocal 6 , STED 7,8 and SIM imaging 9 , but ensuring the quality of the extended pattern 49 across a larger FOV remains a limiting factor. For SMLM, flat-fielding approaches made it 50 possible to extend super-resolution imaging over ~100x100 µm 2 FOVs 10,11 , but their transfer 51 to patterned illumination remains limited. 52An entirely different approach to effectively achieve super-resolution modifies 53 sample preparation rather than image acquisition. By physically increasing the size of the 54 specimen in an isotropic manner via expansion microscopy 12 (ExM), overall imaging 55 throughput can be increased by opting for faster diffraction-limited microscopes 12-14 . 56 Alternatively, combining ExM with existing super-resolution microscopies allows even 57 further improvement in resolution [14][15][16][17][18] , since the resolvable scale is reduced by the 58 expansion factor. However, expansion exacerbates limita...

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Section: Resultsmentioning

confidence: 99%

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Mahecic

Gambarotto

Douglass

et al. 2020

Preprint

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“…Holograms were calculated using an adapted Direct Search (DS) approach, optimising the hologram on a pixel‐by‐pixel basis (Görlitz et al ., ). Computationally, the amplitude at any point in the focal plane

ε (\overset{2false ⇀}{r})

can be calculated as the sum of the contributions from the N pixels in the plane of the SLM (Curtis et al ., ):

\begin{matrix} true \\ right ε false(\overset{⇀}{r} false) & = & left \sum_{i = 0}^{N} A_{0} (\overset{⇀}{ρ_{i}}) exp (i ϕ (\overset{⇀}{ρ_{i}})) K (\overset{2false ⇀}{r}, \overset{2false ⇀}{ρ_{i}}) \\ left prefixexp ((), - i \frac{2 π \overset{⇀}{r} \cdot \overset{⇀}{ρ_{i}}}{λ f}), \end{matrix}

where

A_{0} false({\overset{2false ⇀}{ρ}}_{i} false)

and

φ ({\overset{⇀}{ρ}}_{i})

denote the amplitude and phase of the light incident on the i th pixel on the SLM at a point

\overset{⇀}{ρ}

and λ is the wavelength of the light and f is focal length of the optical train.…”

Section: Methodsmentioning

confidence: 99%

“…Holograms were calculated using an adapted Direct Search (DS) approach, optimising the hologram on a pixel-by-pixel basis (Görlitz et al, 2018). Computationally, the amplitude at any point in the focal plane ( r ) can be calculated as the sum of the contributions from the N pixels in the plane of the SLM (Curtis et al, 2002):…”

Section: Hologram Calculationmentioning

confidence: 99%

Holographic projection for multispot structured illumination microscopy

Ward

Pál

2019

Journal of Microscopy

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Summary This paper describes the design and testing of a multispot structured illumination microscopy system using computer‐generated holograms to create the required excitation patterns. Furthermore, it demonstrates the use of an adapted direct search algorithm for calculating the holograms that allows for imaging across an extended field of view. The system was tested on fixed targets and live cells yielding a two times resolution increase over conventional diffraction‐limited imaging. Lay Description We present the design and testing of a multispot structured illumination microscopy system using computer‐generated holograms to create the required excitation patterns. It demonstrates the use of an adapted direct search algorithm for calculating the holograms that allows for imaging across an extended field of view. The system was tested on fixed targets and live cells yielding a two times resolution increase over conventional diffraction‐limited imaging. The results here demonstrate that holography provides an efficient means of pattern projection for MSIM imaging. It provides a significant improvement in the efficiency of pattern projection and more importantly it allows for the testing of more diverse excitation patterns than possible with amplitude‐only projection. For example, PSF engineering using phase modulation can be easily incorporated into the calculated holograms, potentially generating subdiffraction structures in the excitation pattern. The ability to incorporate PSF engineering into SIM opens up holographic MSIM as a potential method for further increasing resolution with little or no change to the imaging system.

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“…Finally, the impact of tilt, which in this situation corresponds to a misalignment between excitation and depletion foci, is useful to be assessed to estimate the impact of chromatic aberrations [27] or thermal and mechanical drift [13]. We found that 2D-STED was the most sensitive to tilt, followed by z-STED and CH-STED, which can be linked to the respective sizes of their central areas, with a smaller central area leading to a higher vulnerability to misalignment (Figure 7(f)).…”

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

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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easySLM‐STED: Stimulated emission depletion microscopy with aberration correction, extended field of view and multiple beam scanning

Cited by 26 publications

References 22 publications

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Homogeneous multifocal excitation for high-throughput super-resolution imaging

Holographic projection for multispot structured illumination microscopy

Background reduction in STED-FCS using coherent-hybrid STED

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