scholarly journals Verifying molecular clusters by 2-color localization microscopy and significance testing

2019 ◽  
Author(s):  
Andreas M. Arnold ◽  
Magdalena C. Schneider ◽  
Christoph Hüsson ◽  
Robert Sablatnig ◽  
Mario Brameshuber ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Sensitivity ◽  
Significance Testing ◽  
Cellular Structures ◽  
Testing Methods ◽  
P Values ◽  
New Approach ◽  
Localization Microscopy ◽  
Statistical Framework ◽  
Single Molecule Localization Microscopy

AbstractWhile single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential could not be fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the analysis of SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach it is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

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 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 Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

2019 ◽  
Author(s):  
Lekha Patel ◽  
Dylan M. Owen ◽  
Edward A.K. Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Diffraction Limit ◽  
Training Data ◽  
Switching Behavior ◽  
Cellular Structures ◽  
Exact Probability ◽  
Localization Microscopy ◽  
Exact Probability Distribution ◽  
Single Molecule Localization Microscopy

AbstractMany recent advancements in single molecule localization microscopy exploit the stochastic photo-switching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging. Modeling the photo-switching behavior of a fluorophore as a continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photo-switching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a dSTORM experiment involving an arbitrary number of molecules. We demonstrate that when training data is available to estimate photo-switching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations.

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245693
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

Single 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 Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

scholarly journals Single-molecule localization microscopy as nonlinear inverse problem

2019 ◽  
Vol 116 (41) ◽  
pp. 20438-20445
Author(s):  
Ji Yu ◽  
Ahmed Elmokadem
Keyword(s):  
Single Molecule ◽  
Computational Cost ◽  
Image Deconvolution ◽  
Fusion Algorithm ◽  
Localization Microscopy ◽  
Estimation Algorithms ◽  
Statistical Framework ◽  
Spatial Coordinates ◽  
Classical Image ◽  
Single Molecule Localization Microscopy

We present a statistical framework to model the spatial distribution of molecules based on a single-molecule localization microscopy (SMLM) dataset. The latter consists of a collection of spatial coordinates and their associated uncertainties. We describe iterative parameter-estimation algorithms based on this framework, as well as a sampling algorithm to numerically evaluate the complete posterior distribution. We demonstrate that the inverse computation can be viewed as a type of image restoration process similar to the classical image deconvolution methods, except that it is performed on SMLM images. We further discuss an application of our statistical framework in the task of particle fusion using SMLM data. We show that the fusion algorithm based on our model outperforms the current state-of-the-art in terms of both accuracy and computational cost.

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth S. Matikonda ◽  
Dominic A. Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Single Molecule ◽  
New Approach ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy
Sign in / Sign up

Export Citation Format

Share Document