scholarly journals Photon yield enhancement of red fluorophores at cryogenic temperatures

2018 ◽  
Author(s):  
Christiaan N. Hulleman ◽  
Weixing Li ◽  
Ingo Gregor ◽  
Bernd Rieger ◽  
Jörg Enderlein
Keyword(s):  
Liquid Nitrogen ◽  
Single Molecule ◽  
Room Temperature ◽  
Fluorescent Dyes ◽  
Super Resolution ◽  
Yield Enhancement ◽  
Cryogenic Temperatures ◽  
Photon Yield ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle Molecule Localization Microscopy has become one of the most successful and widely applied methods of Super-resolution Fluorescence Microscopy. Its achievable resolution strongly depends on the number of detectable photons from a single molecule until photobleaching. By cooling a sample from room temperature down to liquid nitrogen temperatures, the photostability of dyes can be enhanced by more than 100 fold, which results in an improvement in localization precision greater than 10 times. Here, we investigate a variety of fluorescent dyes in the red spectral region, and we find an average photon yield between 3.5 · 106to 11 · 106photons before bleaching at liquid nitrogen temperatures, corresponding to a theoretical localization precision around 0.1 nm.

scholarly journals Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sheng Liu ◽  
Fang Huang
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Super Resolution ◽  
Resolving Power ◽  
Localization Algorithm ◽  
Whole Cells ◽  
4Pi Microscopy ◽  
Axial Dimension ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractOver the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combined with single-molecule detection/localization and adaptive optics, current 4Pi microscopy methods enabled 10–15 nm isotropic 3D resolution throughout whole cells. However, further improving the achieved 3D resolution poses challenges arising from the complexity of single-molecule emission patterns generated by these coherent single-molecule imaging systems. These complex emission patterns render a large portion of information carrying photons unusable. Here, we introduce a localization algorithm that achieves the theoretical precision limit for a 4Pi based single-molecule switching nanoscopy (4Pi-SMSN) system, and demonstrate improvements in localization precision, accuracy as well as stability comparing with state-of-the-art 4Pi-SMSN methods.

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 Sub-Nanometer Precision using Bayesian Grouping of Localizations

2019 ◽  
Author(s):  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Bernd Rieger ◽  
Ralf Jungmann ◽  
Keith A. Lidke
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Time Course ◽  
Bayesian Method ◽  
A Priori ◽  
Super Resolution ◽  
True Number ◽  
Localization Precision ◽  
Better Than ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.

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.

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

2021 ◽  
Author(s):  
Anders K Engdahl ◽  
Oleg Grauberger ◽  
Mark Schüttpelz ◽  
Thomas Huser
Keyword(s):  
Single Molecule ◽  
Fluorescent Dyes ◽  
Sodium Sulfite ◽  
Super Resolution ◽  
Oxygen Scavenger ◽  
Dark State ◽  
Alexa Fluor ◽  
High Illumination ◽  
Human Osteosarcoma Cells ◽  
Super Resolution Microscopy

Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells.

scholarly journals Super-resolution fight club: assessment of 2D and 3D single-molecule localization microscopy software

2019 ◽  
Vol 16 (5) ◽  
pp. 387-395 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Fight Club ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

Research on DC Breakdown and Flashover Characteristics of PI with Different Temperature

2016 ◽  
Vol 709 ◽  
pp. 11-14
Author(s):  
Tian Ye Niu ◽  
Jia Xin Wu ◽  
Ying Wen Li ◽  
Dong Sheng Xu ◽  
Lu Li ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Rapid Development ◽  
Liquid Nitrogen Temperature ◽  
Power Equipment ◽  
Electrical Characteristics ◽  
Cryogenic Temperatures ◽  
Insulating Materials ◽  
Dielectric Performance ◽  
Working Performance

The electrical characteristics of insulating materials play a key role on the working performance and operation reliability of power equipment. With the rapid development of superconducting technology in recent years,the working temperature of high temperature superconducting power equipment can be controlled around the liquid nitrogen temperature. Due to its excellent dielectric performance and mechanical properties, polyimide have been widely used in power equipment at room temperature. However, polyimide, as a kind of cryogenic insulating materials, is rarely reported at present. Therefore, the study of the insulating characteristics of polyimide at the cryogenic temperatures is of great significance. The DC breakdown property and flashover performance of polyimide are tested around room temperature (300K) and liquid nitrogen temperature (78K). The results show that temperature has some effects on the DC breakdown property and flashover performance of polyimide.

scholarly journals Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

2018 ◽  
Author(s):  
Daniel Sage ◽  
Thanh-An Pham ◽  
Hazen Babcock ◽  
Tomas Lukes ◽  
Thomas Pengo ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Super Resolution ◽  
Large Set ◽  
Holistic View ◽  
Localization Microscopy ◽  
Fight Club ◽  
Community Effort ◽  
2D And 3D ◽  
Single Molecule Localization Microscopy

ABSTRACTWith the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities – 2D, astigmatic 3D, biplane 3D, and double helix 3D – and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

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.

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