scholarly journals A spectral demixing method for high-precision multi-color localization microscopy

2021 ◽  
Author(s):  
Leonid Andronov ◽  
Rachel Genthial ◽  
Didier Hentsch ◽  
Bruno P Klaholz
Keyword(s):  
Single Molecule ◽  
Nuclear Pore ◽  
Drift Correction ◽  
General Applicability ◽  
Localization Microscopy ◽  
Simultaneous Imaging ◽  
Chromatic Aberrations ◽  
Localization Precision ◽  
The Individual ◽  
Single Molecule Localization Microscopy

Single molecule localization microscopy (SMLM) with a dichroic image splitter can provide invaluable multi-color information regarding colocalization of individual molecules, but it often suffers from technical limitations. So far, demixing algorithms give suboptimal results in terms of localization precision and correction of chromatic aberrations. Here we present an image splitter based multi-color SMLM method (splitSMLM) that offers much improved localization precision & drift correction, compensation of chromatic aberrations, and optimized performance of fluorophores in a specific buffer to equalize their reactivation rates for simultaneous imaging. A novel spectral demixing algorithm, SplitViSu, fully preserves localization precision with essentially no data loss and corrects chromatic aberrations at the nanometer scale. Multi-color performance is further improved by using optimized fluorophore and filter combinations. Applied to three-color imaging of the nuclear pore complex (NPC), this method provides a refined positioning of the individual NPC proteins and reveals that Pom121 clusters act as NPC deposition loci, hence illustrating strength and general applicability of the method.

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 A Mean Shift Algorithm for Drift Correction in Localization Microscopy

2021 ◽  
Author(s):  
Frank J Fazekas ◽  
Thomas R Shaw ◽  
Sumin Kim ◽  
Ryan A Bogucki ◽  
Sarah L Veatch
Keyword(s):  
Single Molecule ◽  
Mean Shift ◽  
Time Lapse ◽  
Drift Correction ◽  
Localization Microscopy ◽  
Mean Shift Algorithm ◽  
Peak Finding ◽  
Localization Precision ◽  
Time Lapse Imaging ◽  
Single Molecule Localization Microscopy

Single molecule localization microscopy (SMLM) techniques transcend the diffraction limit of visible light by localizing isolated emitters sampled stochastically. This time-lapse imaging necessitates long acquisition times, over which sample drift can become large relative to the localization precision. Here we present a novel, efficient, and robust method for estimating drift using a simple peak-finding algorithm based on mean shifts that is effective for SMLM in 2 or 3 dimensions.

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 FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

2020 ◽  
Vol 16 (12) ◽  
pp. e1008479
Author(s):  
Daniel F. Nino ◽  
Daniel Djayakarsana ◽  
Joshua N. Milstein
Keyword(s):  
Single Molecule ◽  
Clustering Algorithm ◽  
Spatial Clustering ◽  
Cluster Complex ◽  
Nuclear Pore ◽  
Data Sets ◽  
Localization Microscopy ◽  
Biological Dataset ◽  
Open Source Software Package ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.

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 Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

2020 ◽  
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. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. 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 FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

2019 ◽  
Author(s):  
D. Nino ◽  
D. Djayakarsana ◽  
J. N. Milstein
Keyword(s):  
Single Molecule ◽  
Software Package ◽  
Clustering Algorithm ◽  
Image Resolution ◽  
Nuclear Pore ◽  
Localization Microscopy ◽  
3 Dimensional ◽  
Highly Efficient ◽  
Human Osteosarcoma Cells ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) yields an image resolution 1-2 orders of magnitude below that of conventional light microscopy, resolving fine details on intracellular structure and macromolecular organization. The massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Density based clustering algorithms, such as DBSCAN, can provide spatial statistics on protein/nucleic acid aggregation or dispersion while explicitly identifying macromolecular clusters. The performance of DBSCAN, however, is typically dependent upon an arbitrary, or at least highly subjective, parametric tuning of the algorithm. Moreover, DBSCAN can be computationally expensive, which makes it arduous to evaluate on large image stacks. This is all the more important in 3-dimensions where there exist limited alternatives for quantifying clustering in SMLM data, and where a 2-dimensional analysis of true 3-dimensional data may give rise to image artefacts. We have developed an open-source software package in Python for both identifying and quantifying spatial clustering in 3-dimensional SMLM datasets. FOCAL3D is an extension of our previously developed, 2-dimensional, grid based clustering algorithm FOCAL. FOCAL3D provides a highly efficient way to spatially cluster SMLM datasets, scaling linearly with the number of localizations, and the algorithmic parameters may be systematically optimized so that the resulting analysis is insensitive to variation over a range of parameter choices. We initially validate the performance and parametric insensitivity of FOCAL3D on simulated datasets, then apply the algorithm to 3-dimensional, astigmatic dSTORM images of the nuclear pore complex in human osteosarcoma cells.The data and software package are available at: http://www.utm.utoronto.ca/milsteinlab/software/

Sign in / Sign up

Export Citation Format

Share Document