Evaluating image resolution in stimulated emission depletion microscopy

Tortarolo, Giorgio; Castello, Marco; Diaspro, Alberto; Koho, Sami; Vicidomini, Giuseppe

doi:10.1364/optica.5.000032

Cited by 95 publications

(94 citation statements)

References 21 publications

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“…The use of 2D test samples for testing lateral resolution of localisation based super-resolution has already been covered extensively in the literature [25,[29][30][31]57]. Our focus on 3D samples and the repurposing of a 2D test sample for establishing qPAINT analysis should help to further establish the use of test samples for this more complex set of SMLM properties and analysis strategies.…”

Section: Discussionmentioning

confidence: 99%

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

Lin

Clowsley

Lutz

et al. 2019

Preprint

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Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time-and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

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Section: Discussionmentioning

confidence: 99%

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

Lin

Clowsley

Lutz

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Thanks to the nature of how images are acquired when scanning the sample using the resonant mirror, each raw image contains a mirror image 67 . Having independently acquired these twin images, they then need to be translated so that there is no drift between them, as it can influence the FRC result 66 . We use the subpixel registration algorithm based on the cross correlation of the images 68 .…”

Section: Noise Problem and Resolution Quantification In Sted Imagingmentioning

confidence: 99%

Numerically enhanced adaptive optics-based 3D STED microscopy for deep-tissue super-resolved imaging

Zdańkowski

Trusiak

McGloin

et al. 2019

Preprint

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In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilising both a high power depletion laser (increasing risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here we demonstrate a custom-built 3D STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating, tissue. We introduce and investigate image denoising by block-matching and collaborative filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well-established Fourier ring correlation metric. Experimental validation of the proposed method is achieved through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under a 80µm thick layer of tissue with lateral and axial resolution of 256nm and 300nm, respectively.

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“…Under high light doses, especially for a STED process in order to achieve high resolution, yet, fluorophores tend to be exposed in high risk of photobleaching. Thus, photobleaching leads to signal reduction or information loss, to some extent, affecting to the image quality which in turn asserts limitation to the further improvement of spatial resolution of STED microscopy (Tortarolo et al, 2018). Because phototoxicity is strictly connected with photobleaching, the decrease of photobleaching effect helps to ameliorate phototoxicity phenomenon.…”

Section: Introductionmentioning

confidence: 99%

Recent research on stimulated emission depletion microscopy for reducing photobleaching

Liu

Wang

et al. 2018

Journal of Microscopy

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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.

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Evaluating image resolution in stimulated emission depletion microscopy

Cited by 95 publications

References 21 publications

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples

Numerically enhanced adaptive optics-based 3D STED microscopy for deep-tissue super-resolved imaging

Recent research on stimulated emission depletion microscopy for reducing photobleaching

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