Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy

Rehman, Sohaib Abdul; Carr, Alexander R.; Lenz, Martin O.; Lee, Steven Frank; O’Holleran, Kevin

doi:10.1364/oe.26.004631

Cited by 6 publications

(4 citation statements)

References 12 publications

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“…When the microlens array is placed in an image plane the microlenses themselves sample the spatial domain. Since this configuration results in a loss of spatial resolution, observed most acutely at the image plane (23), the microlens array is optimally located in a plane conjugate to the pupil of the microscope objective for SMLFM (24). This configuration is known as Fourier light field microscopy (25)(26)(27).…”

Section: Light Field Microscopymentioning

confidence: 99%

Single Molecule Light Field Microscopy

Sims

Rehman

Lenz

et al. 2020

Preprint

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We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 µm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by postprocessing 2D localization data generated by common, widelyused, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

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Section: Light Field Microscopymentioning

confidence: 99%

Single Molecule Light Field Microscopy

Sims

Rehman

Lenz

et al. 2020

Preprint

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“…The axial position of the MLA was determined by sending in collimated light through the microscope objective -corresponding to a focused beam in the BFP. The microlens array was placed in this plane as per [14].…”

Section: Mla Alignment For Smlfmmentioning

confidence: 99%

Supplementary document for Single Molecule Light Field Microscopy - 4736529.pdf

Sims,

Rehman,

Lenz

et al. 2020

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This document provides supplementary information to "Single Molecule Light Field Microscopy." 3D POINT SOURCE LOCALIZATION IN SINGLE MOLECULE LIGHT FIELD MICROSCOPYIdeally, a microscope objective transforms a spherical wave emitted by a point source located at the origin x p , y p , z p into a plane wave at the pupil. Spherical waves emitted by a point emitter displaced from the origin, located at (x i , y i , z i ), generate more complex wavefronts in the pupil since in this case the optical path length to the principal sphere varies as a function of propagation direction. The majority of microscope objectives are designed according to the sine condition, according to which, ray height is preserved during mapping between principal planes ({x p , y p } → {u, v}) [11]. As a result, the phase difference in the pupil between wavefronts emanating from points located at x p , y p , z p and (x i , y i , z i ) depends on the optical path difference (OPD) between two rays that intersect at the principal sphere:

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“…Sub-diffraction axial precision can be achieved by engineering the shape of the PSF to simultaneously encode the lateral and axial position of a single emitter in a 2D image 13 , 14 . A variety of engineered PSFs have been reported, including astigmatism ( ~ 1 μm DoF) 15 , a bisected pupil ( ~ 1 μm DoF) 16 , the corkscrew PSF ( ~ 3 μm DoF) 17 , the double helix PSF ( ~ 4 μm DoF) 5 , 18 , 19 , and the tetrapod PSF (6–20 μm DoF) 20 , 21 .…”

Section: Introductionmentioning

confidence: 99%

High-density volumetric super-resolution microscopy

Daly,

Ferreira Fernandes,

Bruggeman

et al. 2024

Nat Commun

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Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We demonstrate this optical robustness experimentally with high accuracy ( > 99.2 ± 0.1%, 0.1 locs μm−2) and sensitivity ( > 86.6 ± 0.9%, 0.1 locs μm−2) through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high-density volumetric imaging (0.15 locs μm−2) in dense cytosolic tubulin datasets.

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Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy

Cited by 6 publications

References 12 publications

Single Molecule Light Field Microscopy

Single Molecule Light Field Microscopy

Supplementary document for Single Molecule Light Field Microscopy - 4736529.pdf

High-density volumetric super-resolution microscopy

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