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

2019 ◽  
Author(s):  
Yiming Li ◽  
Yu-Le Wu ◽  
Philipp Hoess ◽  
Markus Mund ◽  
Jonas Ries
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Software Tool ◽  
Calibration Procedure ◽  
Experimental Calibration ◽  
Point Spread ◽  
Processing Step ◽  
Spread Function ◽  
The Individual ◽  
Single Molecule Localization Microscopy

Abstract3D 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 z-localization 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.

scholarly journals Photon count estimation in single-molecule localization microscopy

2018 ◽  
Author(s):  
Rasmus Ø. Thorsen ◽  
Christiaan N. Hulleman ◽  
Mathias Hammer ◽  
David Grünwald ◽  
Sjoerd Stallinga ◽  
...  
Keyword(s):  
Single Molecule ◽  
Axial Position ◽  
Optical Aberrations ◽  
Intensity Estimation ◽  
Localization Microscopy ◽  
Photon Count ◽  
Point Spread ◽  
Spread Function ◽  
Gaussian Point ◽  
Single Molecule Localization Microscopy

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.

scholarly journals Information-rich localization microscopy through machine learning

2018 ◽  
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.

scholarly journals Phasor based single-molecule localization microscopy in 3D (pSMLM-3D): an algorithm for MHz localization rates using standard CPUs

2017 ◽  
Author(s):  
Koen J.A. Martens ◽  
Arjen N. Bader ◽  
Sander Baas ◽  
Bernd Rieger ◽  
Johannes Hohlbein
Keyword(s):  
Single Molecule ◽  
Iterative Algorithms ◽  
Region Of Interest ◽  
Depth Information ◽  
Localization Algorithm ◽  
Two Phase ◽  
Model Free ◽  
Spread Function ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

AbstractWe present a fast and model-free 2D and 3D single-molecule localization algorithm that allows more than 3 million localizations per second on a standard multi-core CPU with localization accuracies in line with the most accurate algorithms currently available. Our algorithm converts the region of interest around a point spread function (PSF) to two phase vectors (phasors) by calculating the first Fourier coefficients in both x- and y-direction. The angles of these phasors are used to localize the center of the single fluorescent emitter, and the ratio of the magnitudes of the two phasors is a measure for astigmatism, which can be used to obtain depth information (z-direction). Our approach can be used both as a stand-alone algorithm for maximizing localization speed and as a first estimator for more time consuming iterative algorithms.

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

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.

scholarly journals Multiplane and Spectrally-Resolved Single Molecule Localization Microscopy with Industrial Grade CMOS Cameras

2017 ◽  
Author(s):  
Hazen P. Babcock
Keyword(s):  
Open Source ◽  
Single Molecule ◽  
Open Source Software ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Industrial Grade ◽  
Spectrally Resolved ◽  
Imaging Plane ◽  
Single Molecule Localization Microscopy

ABSTRACTIn this work we explore the use of industrial grade CMOS cameras for single molecule localization microscopy (SMLM). We show that the performance of these cameras in single imaging plane SMLM applications is comparable to much more expensive scientific CMOS (sCMOS) cameras. We show that these cameras can be used in more demanding biplane, multiplane and spectrally resolved SMLM applications. The 10-40× reduction in camera cost makes it practical to build SMLM setups with 4 or more cameras. In addition we provide open-source software for simultaneously controlling multiple CMOS cameras and for the reduction of the movies that are acquired to super-resolution images.

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