scholarly journals Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

2021 ◽  
Author(s):  
Mark Bates ◽  
Jan Keller-Findeisen ◽  
Adrian Przybylski ◽  
Andreas Hüper ◽  
Till Stephan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Optical System ◽  
Temporal Dynamics ◽  
Optical Design ◽  
Primary Neurons ◽  
Mammalian Mitochondria ◽  
Spread Function ◽  
Localization Precision ◽  
Image Detail ◽  
Single Molecule Localization Microscopy

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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 Improved single-molecule localization precision in astigmatism-based 3D superresolution imaging using weighted likelihood estimation

2018 ◽  
Author(s):  
Christopher H. Bohrer ◽  
Xinxing Yang ◽  
Zhixin Lyu ◽  
Shih-Chin Wang ◽  
Jie Xiao
Keyword(s):  
Single Molecule ◽  
Optical System ◽  
Gaussian Function ◽  
Gaussian Model ◽  
Likelihood Estimation ◽  
Three Dimensions ◽  
Weighted Likelihood ◽  
Superresolution Microscopy ◽  
Engineering Technique ◽  
Localization Precision

AbstractAstigmatism-based superresolution microscopy is widely used to determine the position of individual fluorescent emitters in three-dimensions (3D) with subdiffraction-limited resolutions. This point spread function (PSF) engineering technique utilizes a cylindrical lens to modify the shape of the PSF and break its symmetry above and below the focal plane. The resulting asymmetric PSFs at different z-positions for single emitters are fit with an elliptical 2D-Gaussian function to extract the widths along two principle x- and y-axes, which are then compared with a pre-measured calibration function to determine its z-position. While conceptually simple and easy to implement, in practice, distorted PSFs due to an imperfect optical system often compromise the localization precision; and it is laborious to optimize a multi-purpose optical system. Here we present a methodology that is independent of obtaining a perfect PSF and enhances the localization precision along the z-axis. By utilizing multiple calibration images of fluorescent beads at varying z-planes and characterizing experimentally measured background distributions, we numerically approximated the probability of observing a certain signal in a given pixel from a single emitter at a particular z-plane. We then used a weighted maximum likelihood estimator (WLE) to determine the 3D-position of the emitter. We demonstrate that this approach enhances z-axis localization precision in all conditions we tested, in particular when the PSFs deviate from a standard 2D Gaussian model.

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 A workflow for sizing oligomeric biomolecules based on cryo single molecule localization microscopy

2020 ◽  
Author(s):  
Magdalena C. Schneider ◽  
Roger Telschow ◽  
Gwenael Mercier ◽  
Montserrat López-Martinez ◽  
Otmar Scherzer ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Reliability ◽  
Limiting Factor ◽  
Dye Molecules ◽  
New Approach ◽  
Localization Microscopy ◽  
Additional Information ◽  
Localization Precision ◽  
Microscopy Data ◽  
Single Molecule Localization Microscopy

ABSTRACTSingle molecule localization microscopy (SMLM) has enormous potential for resolving subcellular structures below the diffraction limit of light microscopy: Localization precision in the low digit nanometer regime has been shown to be achievable. In order to record localization microscopy data, however, sample fixation is inevitable to prevent molecular motion during the rather long recording times of minutes up to hours. Eventually, it turns out that preservation of the sample’s ultrastructure during fixation becomes the limiting factor. We propose here a workflow for data analysis, which is based on SMLM performed at cryogenic temperatures. Since molecular dipoles of the fluorophores are fixed at low temperatures, such an approach offers the possibility to use the orientation of the dipole as an additional information for image analysis. In particular, assignment of localizations to individual dye molecules becomes possible with high reliability. We quantitatively characterized the new approach based on the analysis of simulated oligomeric structures. Side lengths can be determined with a relative error of less than 1% for tetramers with a nominal side length of 5 nm, even if the assumed localization precision for single molecules is more than 2 nm.

scholarly journals Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

2020 ◽  
Vol 6 (16) ◽  
pp. eaay8271 ◽  
Author(s):  
Simao Coelho ◽  
Jongho Baek ◽  
Matthew S. Graus ◽  
James M. Halstead ◽  
Philip R. Nicovich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Distance Measurements ◽  
Intact Cells ◽  
Localization Microscopy ◽  
Direct Distance ◽  
Time Drift ◽  
Localization Precision ◽  
20 Nm ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

scholarly journals Direct supercritical angle localization microscopy for nanometer 3D superresolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anindita Dasgupta ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
Uwe Hübner ◽  
Jan Becker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
State Of The Art ◽  
Dna Origami ◽  
Cellular Structures ◽  
Full Potential ◽  
Localization Microscopy ◽  
Structural Cell ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

scholarly journals Improved localization precision via restricting confined biomolecule stochastic motion in single-molecule localization microscopy

Nanophotonics ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jielei Ni ◽  
Bo Cao ◽  
Gang Niu ◽  
Danni Chen ◽  
Guotao Liang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Direct Evidence ◽  
Diffraction Limit ◽  
Drift Correction ◽  
Stochastic Motion ◽  
Localization Microscopy ◽  
Alexa Fluor ◽  
Biological Studies ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract Single-molecule localization microscopy (SMLM) plays an irreplaceable role in biological studies, in which nanometer-sized biomolecules are hardly to be resolved due to diffraction limit unless being stochastically activated and accurately located by SMLM. For biological samples preimmobilized for SMLM, most biomolecules are cross-linked and constrained at their immobilizing sites but still expected to undergo confined stochastic motion in regard to their nanometer sizes. However, few lines of direct evidence have been reported about the detectability and influence of confined biomolecule stochastic motion on localization precision in SMLM. Here, we access the potential stochastic motion for each immobilized single biomolecule by calculating the displacements between any two of its localizations at different frames during sequential imaging of Alexa Fluor-647-conjugated oligonucleotides. For most molecules, localization displacements are remarkably larger at random frame intervals than at shortest intervals even after sample drift correction, increase with interval times and then saturate, showing that biomolecule stochastic motion is detected and confined around the immobilizing sizes in SMLM. Moreover, localization precision is inversely proportional to confined biomolecule stochastic motion, whereas it can be deteriorated or improved by enlarging the biomolecules or adding a post-crosslinking step, respectively. Consistently, post-crosslinking of cell samples sparsely stained for tubulin proteins results in a better localization precision. Overall, this study reveals that confined stochastic motion of immobilized biomolecules worsens localization precision in SMLM, and improved localization precision can be achieved via restricting such a motion.

scholarly journals Combining 3D single molecule localization strategies for reproducible bioimaging

2018 ◽  
Author(s):  
Clément Cabriel ◽  
Nicolas Bourg ◽  
Pierre Jouchet ◽  
Guillaume Dupuis ◽  
Christophe Leterrier ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
3D Localization ◽  
Point Spread ◽  
Spread Function ◽  
Multicolor Imaging ◽  
Localization Precision ◽  
Different Depths ◽  
Super Resolution Microscopy ◽  
Living Bacteria

We developed a 3D localization-based super-resolution technique providing a slowly varying localization precision 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 to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive 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.

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