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

2019 ◽  
Vol 10 (18) ◽  
pp. 4914-4922 ◽  
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.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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

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.

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

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.

Visualization and intracellular dynamic tracking of exosomes and exosomal miRNAs using single molecule localization microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 5154-5162 ◽  
Author(s):  
Chen Chen ◽  
Shenfei Zong ◽  
Zhuyuan Wang ◽  
Ju Lu ◽  
Dan Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Dynamic Tracking ◽  
Exosomal Mirnas ◽  
Resolution Imaging ◽  
Intracellular Dynamic ◽  
Single Molecule Localization Microscopy

Super-resolution imaging and dynamic tracking of cancer-derived exosomes and exosomal miRNAs were realized using single molecule localization microscopy.

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