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

Recently, Franke, Sauer and van de Linde1 introduced a way to estimate the axial position of single-molecules (TRABI). To this end, they compared the detected photon count from a temporal radial-aperture-based intensity estimation to the estimated count from Gaussian point-spread function (PSF) fitting to the data. Empirically they found this photometric ratio to be around 0.7-0.8 close to focus and decreasing away from it. Here, we explain this reported but unexplained discrepancy and furthermore show that the photometric ratio as indicator for axial position is susceptible even to typical optical aberrations.

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

10.1101/2021.09.13.460135 ◽

2021 ◽

Author(s):

Tianben Ding ◽

Matthew D. Lew

Keyword(s):

Lipid Bilayers ◽

Single Molecule ◽

Point Spread Function ◽

Soft Matter ◽

Rotational Diffusion ◽

Nile Red ◽

Emission Anisotropy ◽

Vortex Point ◽

Point Spread ◽

Spread Function

AbstractWithin 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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Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

10.1101/2020.10.01.322834 ◽

2020 ◽

Author(s):

Christiaan N. Hulleman ◽

Rasmus Ø. Thorsen ◽

Sjoerd Stallinga ◽

Bernd Rieger

Keyword(s):

Single Molecule ◽

Point Spread Function ◽

Polar Angle ◽

Phase Mask ◽

Simultaneous Estimation ◽

Dipole Orientation ◽

Localization Microscopy ◽

Point Spread ◽

Spread Function ◽

Microscopy Techniques

We have developed an engineered Point Spread Function (PSF) to enable the simultaneous estimation of dipole orientation, 3D position and degree of rotational constraint of single-molecule emitters from a single 2D focal plane. Besides giving access to orientation information, the Vortex PSF along with the vectorial PSF fitter avoids localization bias common in localization microscopy for fixed dipole emitters. We demonstrate this technique on reorienting single-molecules and using binding-activated localization microscopy on DNA intercalators, corroborating perpendicular azimuthal angles to the DNA axis for in-plane emitters but find a non-uniform distribution as a function of the polar angle. The Vortex PSF is realized by an affordable glass phase mask and has a compact footprint that can easily be combined with localization microscopy techniques on rotationally constrained emitters.

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Information-rich localization microscopy through machine learning

10.1101/373878 ◽

2018 ◽

Cited By ~ 1

Author(s):

Taehwan Kim ◽

Seonah Moon ◽

Ke Xu

Keyword(s):

Single Molecule ◽

Data Driven ◽

Direct Link ◽

Localization Microscopy ◽

Point Spread ◽

Spread Function ◽

Data Driven Approach ◽

Multidimensional Information ◽

Specific Alteration ◽

Single Molecule Localization Microscopy

While current single-molecule localization microscopy (SMLM) methods often rely on the target-specific alteration of the point spread function (PSF) to encode the multidimensional contents of single fluorophores, we argue that the details of the PSF in an unmodified microscope already contain rich, multidimensional information. We introduce a data-driven approach in which artificial neural networks (ANNs) are trained to make a direct link between an experimental PSF image and its underlying parameters. To demonstrate this concept in real systems, we decipher in fixed cells both the colors and the axial positions of single molecules in regular SMLM data.

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Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Nature Communications ◽

10.1038/s41467-021-26228-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Christiaan N. Hulleman ◽

Rasmus Ø. Thorsen ◽

Eugene Kim ◽

Cees Dekker ◽

Sjoerd Stallinga ◽

...

Keyword(s):

Single Molecule ◽

Point Spread Function ◽

Position Estimation ◽

Phase Plate ◽

Light Path ◽

Excitation Light ◽

Localization Microscopy ◽

Point Spread ◽

Spread Function ◽

Polarization Splitting

AbstractEstimating the orientation and 3D position of rotationally constrained emitters with localization microscopy typically requires polarization splitting or a large engineered Point Spread Function (PSF). Here we utilize a compact modified PSF for single molecule emitter imaging to estimate simultaneously the 3D position, dipole orientation, and degree of rotational constraint from a single 2D image. We use an affordable and commonly available phase plate, normally used for STED microscopy in the excitation light path, to alter the PSF in the emission light path. This resulting Vortex PSF does not require polarization splitting and has a compact PSF size, making it easy to implement and combine with localization microscopy techniques. In addition to a vectorial PSF fitting routine we calibrate for field-dependent aberrations which enables orientation and position estimation within 30% of the Cramér-Rao bound limit over a 66 μm field of view. We demonstrate this technique on reorienting single molecules adhered to the cover slip, λ-DNA with DNA intercalators using binding-activated localization microscopy, and we reveal periodicity on intertwined structures on supercoiled DNA.

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