Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation

Curcio, Valentina; Alemán-Castañeda, Luis A.; Brown, Thomas G.; Brasselet, Sophie; Alonso, Miguel A.

doi:10.1038/s41467-020-19064-6

Cited by 61 publications

(75 citation statements)

References 46 publications

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“…The polarized vortex PSF clearly resolves freely rotating molecules (Figure 1C) from fixed molecules (Figure 1D), as well as an SM wobbling largely in the α direction (Figure 1E) vs. one wobbling mostly in the β direction (Figure 1F). In fact, the polarized vortex PSF provides superior precision for measuring the second moments m compared to several state-of-the-art orientation-sensing PSFs, including the polarized standard PSF, 10 bifocal microscope, 45 CHIDO, 21 and unpolarized vortex PSF. 23 This performance holds when molecules are near a refractive index interface as well as when they are within a matched medium (Figure S3).…”

Section: Image Formation and The Polarized Vortex Psfmentioning

confidence: 99%

“…4,10,11 The field continues to evolve with theo-retical studies to determine fundamental performance limits, [15][16][17] the assistance of machine learning to design optimal PSFs for 3D SMLM, [18][19][20] and new PSFs for measuring the 3D positions and 3D orientations of SMs. [21][22][23] The central challenge facing SM spectroscopists is the finite photon budget afforded to each individual fluorophore before irreversible photobleaching; due to photon shot noise, it is difficult to measure experimentally the many properties of SM emission, e.g., the position, arrival time, wavelength, and polarization of each incoming photon, simultaneously with high precision. Tradeoffs are unavoidable, and one's sensitivity to a particular parameter can be improved to incredible degrees by reducing sensitivity to others, e.g., achieving~1 nm localization precision in MINFLUX by making the measurement largely independent of emission wavelength and polarization.…”

Section: Introductionmentioning

confidence: 99%

“…Second, existing techniques are largely limited to estimating rotational diffusion, or the "wobble" of an SM, as a scalar parameter averaged over all directions. 16,17,21,23,[25][26][27] However, we know molecules wobble in full 3D space and, in certain environments, perhaps anisotropically, i.e., more in one direction versus another. 28,29 Is it possible to robustly quantify the degree of wobble anisotropy of an SM experimentally?…”

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Ding

Lew

2021

Preprint

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Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule's wobble, simultaneously from a single image. Even when imaging dim emitters (~500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8° orientation precision, and 26° wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive using molecular positions alone.

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Section: Image Formation and The Polarized Vortex Psfmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Ding

Lew

2021

Preprint

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“…More recently, polarized two-channel imaging with separated x-y localization has been proposed to mitigate the problem [ 54 ]. Furthermore, recent advances have been made towards the joint estimation of position, orientation and wobbling parameters using polarized four channel detection [ 55 – 57 ].…”

Section: Systematic Errors Caused By Fixed Dipole Orientationsmentioning

confidence: 99%

Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence

Zelger¹,

Bodner²,

Offterdinger³

et al. 2021

Biomed. Opt. Express

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The precise spatial localization of single molecules in three dimensions is an important basis for single molecule localization microscopy (SMLM) and tracking. At distances up to a few hundred nanometers from the coverslip, evanescent wave coupling into the glass, also known as supercritical angle fluorescence (SAF), can strongly improve the axial precision, thus facilitating almost isotropic localization performance. Specific detection systems, introduced as Supercritical angle localization microscopy (SALM) or Direct optical nanoscopy with axially localized detection (DONALD), have been developed to exploit SAF in modified two-channel imaging schemes. Recently, our group has shown that off-focus microscopy, i.e., imaging at an intentional slight defocus, can perform equally well, but uses only a single detection arm. Here we compare SALM, off-focus imaging and the most commonly used 3D SMLM techniques, namely cylindrical lens and biplane imaging, regarding 3D localization in close proximity to the coverslip. We show that all methods gain from SAF, which leaves a high detection NA as the only major key requirement to unlock the SAF benefit. We find parameter settings for cylindrical lens and biplane imaging for highest z-precision. Further, we compare the methods in view of robustness to aberrations, fixed dipole emission and double-emitter events. We show that biplane imaging provides the best overall performance and support our findings by DNA-PAINT experiments on DNA-nanoruler samples. Our study sheds light on the effects of SAF for SMLM and is helpful for researchers who plan to employ localization-based 3D nanoscopy close to the coverslip.

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“…The development of an optimal method to disentangle spatial position, mean orientation and orientation fluctuations is still an ongoing research 13 . One approach is to encode the orientation and wobbling information into the shape of the single molecules’ point spread function (PSF), using custom-designed phase or birefringent masks 14,15,16 . This strategy comes however at the price of some constraints, which restrain its use in regular SMLM imaging.…”

Section: Introductionmentioning

confidence: 99%

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

Rimoli

Cav

Curcio

et al. 2021

Preprint

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Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context.

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Birefringent Fourier filtering for single molecule coordinate and height super-resolution imaging with dithering and orientation

Cited by 61 publications

References 46 publications

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble using a Polarized Vortex Point Spread Function

Three-dimensional single molecule localization close to the coverslip: a comparison of methods exploiting supercritical angle fluorescence

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

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