Aberration-accounting calibration for 3D single-molecule localization microscopy

Cabriel, Clément; Bourg, Nicolas; Dupuis, Guillaume; Lévêque‐Fort, Sandrine

doi:10.1364/ol.43.000174

Cited by 37 publications

(37 citation statements)

References 25 publications

(28 reference statements)

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“…with a fixed focus plane and dSTORM fluorophores. More specifically, we used a sample of 15 µm microspheres decorated with fluorophores (either AF647 or AF555), as described in [20]. By measuring the position of the center and the radius of the spheres, it is possible to calculate the expected axial position of each molecule from the measurement of its lateral position.…”

Section: Methodsmentioning

confidence: 99%

“…As a result, this technique, called Dual-view Astigmatic Imaging with SAF Yield (DAISY), exhibits a weakly anisotropic resolution over the whole capture range. We first performed the calibration of the astigmatism-based axial detection using 15 µm diameter latex microspheres coated with Alexa Fluor (AF) 647 as described in [20] in order to account for the influence of the optical aberrations on the PSFs and thus eliminate this axial bias source (see Methods). Then, to evaluate the localization precision of DAISY, we imaged dark red 40-nm diameter fluorescent beads located at various randomly distributed heights with a weak 637 nm excitation so that their emission level matched that of AF647 in typical dSTORM conditions, i.e.…”

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confidence: 99%

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Combining 3D single molecule localization strategies for reproducible bioimaging

Cabriel

Bourg

Jouchet

et al. 2018

Preprint

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We developed a 3D localization-based super-resolution technique providing an almost isotropic 3D resolution over a 1 µm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tiltinsensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several µm range by direct merging of multiple acquisitions at different depths.Despite recent advances in localization-based super-resolution techniques, 3D fluorescence imaging of biological samples remains a major challenge, mostly because of its lack of versatility. While photoactivated localization microscopy (PALM) and (direct) stochastic optical reconstruction microscopy ((d)STORM) can easily provide lateral a localization precision (i.e. the standard deviation of the position) down to 5-10 nm [1,2,3,4], a great deal of effort is being made to develop quantitative and reproducible 3D super-localization methods. The most widely used 3D SMLM technique is the astigmatic imaging, which relies on the use of a cylindrical lens to apply an astigmatic aberration in the detection path to encode the axial information in the shape of the spots, achieving an axial localization precision down to 20-25 nm [5]-though the precision varies with the axial position. Other Point Spread Function (PSF) shaping methods are also available [6,7,8], but their implementations are not as inexpensive and straightforward. Still, all PSF shaping methods including astigmatic imaging suffer from several drawbacks such as axial drifts, chromatic aberration, field-varying geometrical aberrations and sample tilts. These sources of biases often degrade the resolution or hinder 1 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/385799 doi: bioRxiv preprint first posted online Aug. 6, 2018; colocalization and experiment reproducibility. Axial measurements can also be performed thanks to intensity-based techniques like Supercritical Angle Fluorescence (SAF) [9,10,11,12,13,14], which relies on the detection of the near-field emission of fluorophores coupled into propagative waves at the sample/glass coverslip interface due to the index mismatch. Combined with SMLM, this technique, called Direct Optical Nanoscopy with Axially Localized ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Combining 3D single molecule localization strategies for reproducible bioimaging

Cabriel

Bourg

Jouchet

et al. 2018

Preprint

Self Cite

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“…A common strategy to generate a depth-dependent PSF calibration is to measure many fluorophores at different distances away from the coverslip to generate a calibration curve. This can be done either by 1) embedding beads in a gel at different heights [15] or 2) attaching fluorophores on a defined curved surface [16]. Then, each fluorescent molecule gives a random sampling of the z-position.…”

Section: Introductionmentioning

confidence: 99%

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

Li¹,

Hoess

et al. 2019

Preprint

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3D Single molecule localization microscopy relies on fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause are depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate zlocalization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

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“…Now, commercial slides with 3D DNA-origami structures are available which provide a stringent test of 3D imaging performance. In addition, we [45] and others [46,47] have established DNA-PAINT samples based on surface coated microspheres which can be used for a variety of testing purposes associated with DNA-PAINT, including their use as convenient 3D structures of known shape.…”

Section: Assessment Of 3d Imaging With a Dna-paint Microsphere Samplementioning

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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Aberration-accounting calibration for 3D single-molecule localization microscopy

Cited by 37 publications

References 25 publications

Combining 3D single molecule localization strategies for reproducible bioimaging

Combining 3D single molecule localization strategies for reproducible bioimaging

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

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

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