Analysis of super-resolution single molecule localization microscopy data: A tutorial

Novel insights and more powerful analytical tools can emerge from the reanalysis of existing data sets, especially via machine learning methods. Despite the widespread use of single molecule localization microscopy (SMLM) for super-resolution bioimaging, the underlying data are often not publicly accessible. We developed ShareLoc (https://shareloc.xyz), an open platform designed to enable sharing, easy visualization and reanalysis of SMLM data. We discuss its features and show how data sharing can improve the performance and robustness of SMLM image reconstruction by deep learning.

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Analyzing Single Molecule Localization Microscopy Data Using Cubic Splines

10.1101/083402 ◽

2016 ◽

Cited By ~ 2

Author(s):

Hazen P. Babcock ◽

Xiaowei Zhuang

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Computation Time ◽

Cubic Splines ◽

Localization Algorithm ◽

Localization Microscopy ◽

Analytical Functions ◽

Open Source Software Package ◽

Microscopy Data ◽

Single Molecule Localization Microscopy

AbstractThe resolution of super-resolution microscopy based on single molecule localization is in part determined by the accuracy of the localization algorithm. In most published approaches to date this localization is done by fitting an analytical function that approximates the point spread function (PSF) of the microscope. However, particularly for localization in 3D, analytical functions such as a Gaussian, which are computationally inexpensive, may not accurately capture the PSF shape leading to reduced fitting accuracy. On the other hand, analytical functions that can accurately capture the PSF shape, such as those based on pupil functions, can be computationally expensive. Here we investigate the use of cubic splines as an alternative fitting approach. We demonstrate that cubic splines can capture the shape of any PSF with high accuracy and that they can be used for fitting the PSF with only a 2-3x increase in computation time as compared to Gaussian fitting. We provide an open-source software package that measures the PSF of any microscope and uses the measured PSF to perform 3D single molecule localization microscopy analysis with reasonable accuracy and speed.

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

10.26434/chemrxiv.14438801 ◽

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>

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Parameter-free rendering of single-molecule localization microscopy data for parameter-free resolution estimation

Communications Biology ◽

10.1038/s42003-021-02086-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Adrien C. Descloux ◽

Kristin S. Grußmayer ◽

Aleksandra Radenovic

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Free Resolution ◽

Temporal Separation ◽

Localization Microscopy ◽

Resolution Imaging ◽

Localization Precision ◽

Microscopy Data ◽

Resolution Estimation ◽

Single Molecule Localization Microscopy

AbstractLocalization microscopy is a super-resolution imaging technique that relies on the spatial and temporal separation of blinking fluorescent emitters. These blinking events can be individually localized with a precision significantly smaller than the classical diffraction limit. This sub-diffraction localization precision is theoretically bounded by the number of photons emitted per molecule and by the sensor noise. These parameters can be estimated from the raw images. Alternatively, the resolution can be estimated from a rendered image of the localizations. Here, we show how the rendering of localization datasets can influence the resolution estimation based on decorrelation analysis. We demonstrate that a modified histogram rendering, termed bilinear histogram, circumvents the biases introduced by Gaussian or standard histogram rendering. We propose a parameter-free processing pipeline and show that the resolution estimation becomes a function of the localization density and the localization precision, on both simulated and state-of-the-art experimental datasets.

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

10.26434/chemrxiv.14438801.v1 ◽

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>

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Quantitative super-resolution single molecule microscopy dataset of YFP-tagged growth factor receptors

10.1101/246488 ◽

2018 ◽

Author(s):

Tomáš Lukeš ◽

Jakub Pospíšil ◽

Karel Fliegel ◽

Theo Lasser ◽

Guy M. Hagen

Keyword(s):

Data Analysis ◽

Growth Factor ◽

Single Molecule ◽

Super Resolution ◽

Growth Factor Receptors ◽

Data Sets ◽

Localization Microscopy ◽

Super Resolution Microscopy ◽

Resolution Single ◽

Single Molecule Localization Microscopy

BackgroundSuper-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared to organic dyes which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms.FindingsFour complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM data sets using a different method: super-resolution optical fluctuation imaging (SOFI). The two modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes.ConclusionThis dataset has potential for extensive reuse. Complete raw data from SMLM experiments has typically not been published. The YFP data exhibits low signal to noise ratios, making data analysis a challenge. These data sets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.

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A H-bond strategy to develop acid-resistant photoswitchable rhodamine spirolactams for super-resolution single-molecule localization microscopy

Chemical Science ◽

10.1039/c9sc01284b ◽

2019 ◽

Vol 10 (18) ◽

pp. 4914-4922 ◽

Cited By ~ 26

Author(s):

Qingkai Qi ◽

Weijie Chi ◽

Yuanyuan Li ◽

Qinglong Qiao ◽

Jie Chen ◽

...

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Amino Groups ◽

Localization Microscopy ◽

Resolution Imaging ◽

Resolution Single ◽

Single Molecule Localization Microscopy

Rhodamine spirolactams with adjacent amino groups work as acid-resistant and photoswitchable fluorophores in single-molecule localization super-resolution imaging.

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