scholarly journals A theoretical high-density nanoscopy study leads to the design of UNLOC, an unsupervised algorithm

2018 ◽  
Author(s):  
Sébastien Mailfert ◽  
Jérôme Touvier ◽  
Lamia Benyoussef ◽  
Roxane Fabre ◽  
Asma Rabaoui ◽  
...  
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Signal To Noise Ratio ◽  
Single Molecules ◽  
High Density ◽  
Superresolution Microscopy ◽  
Central Interest ◽  
Fluorescent Particles ◽  
Particle Localization ◽  
Cramer Rao Bound

ABSTRACTAmong the superresolution microscopy techniques, the ones based on serially imaging sparse fluorescent particles enable the reconstruction of high-resolution images by localizing single molecules. Although challenging, single-molecule localization microscopy (SMLM) methods aim at listing the position of individual molecules leading a proper quantification of the stoichiometry and spatial organization of molecular actors. However, reaching the precision requested to localize accurately single molecules is mainly constrained by the signal-to-noise ratio (SNR) but also the density (Dframe), i.e., the number of fluorescent particles per μm2 per frame. Of central interest, we establish here a comprehensive theoretical study relying on both SNR and Dframe to delineate the achievable limits for accurate SMLM observations. We demonstrate that, for low-density hypothesis (i.e. one-Gaussian fitting hypothesis), any fluorescent particle biases the localization of a particle of interest when they are distant by less than ≈ 600 nm. Unexpectedly, we also report that even dim fluorescent particles should be taken into account to ascertain unbiased localization of any surrounding particles. Therefore, increased Dframe quickly deteriorates the localization precision, the image reconstruction and more generally the quantification accuracy. The first outcome is a standardized density-SNR space diagram to determine the achievable SMLM resolution expected with experimental data. Additionally, this study leads to the identification of the essential requirements for implementing UNLOC (UNsupervised particle LOCalization), an unsupervised and fast computing algorithm approaching the Cramér-Rao bound for particles at high-density per frame and without any prior on their intensity. UNLOC is available as an ImageJ plugin.

scholarly journals Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

2015 ◽  
Vol 112 (32) ◽  
pp. E4390-E4399 ◽  
Author(s):  
Mathew Stracy ◽  
Christian Lesterlin ◽  
Federico Garza de Leon ◽  
Stephan Uphoff ◽  
Pawel Zawadzki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Spatial Organization ◽  
Search Time ◽  
Population Level ◽  
Bacterial Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

scholarly journals Particle localization using local gradients and its application to nanometer stabilization of a microscope

2021 ◽  
Author(s):  
Anatolii V. Kashchuk ◽  
Oleksandr Perederiy ◽  
Chiara Caldini ◽  
Lucia Gardini ◽  
Francesco Saverio Pavone ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Universal Method ◽  
Superresolution Microscopy ◽  
Accurate Localization ◽  
Micro Particles ◽  
Localization Algorithms ◽  
Gradient Calculation ◽  
Particle Localization ◽  
Local Gradient

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

scholarly journals Single-molecule optical absorption imaging by nanomechanical photothermal sensing

2018 ◽  
Vol 115 (44) ◽  
pp. 11150-11155 ◽  
Author(s):  
Miao-Hsuan Chien ◽  
Mario Brameshuber ◽  
Benedikt K. Rossboth ◽  
Gerhard J. Schütz ◽  
Silvan Schmid
Keyword(s):  
Single Molecule ◽  
Shot Noise ◽  
Signal To Noise Ratio ◽  
Local Heating ◽  
Single Molecules ◽  
High Sensitivity ◽  
Temperature Sensitive ◽  
Signal To Noise ◽  
Promising Alternative ◽  
Noise Ratio

Absorption microscopy is a promising alternative to fluorescence microscopy for single-molecule imaging. So far, molecular absorption has been probed optically via the attenuation of a probing laser or via photothermal effects. The sensitivity of optical probing is not only restricted by background scattering but it is fundamentally limited by laser shot noise, which minimizes the achievable single-molecule signal-to-noise ratio. Here, we present nanomechanical photothermal microscopy, which overcomes the scattering and shot-noise limit by detecting the photothermal heating of the sample directly with a temperature-sensitive substrate. We use nanomechanical silicon nitride drums, whose resonant frequency detunes with local heating. Individual Au nanoparticles with diameters from 10 to 200 nm and single molecules (Atto 633) are scanned with a heating laser with a peak irradiance of 354 ± 45 µW/µm2 using 50× long-working-distance objective. With a stress-optimized drum we reach a sensitivity of 16 fW/Hz1/2 at room temperature, resulting in a single-molecule signal-to-noise ratio of >70. The high sensitivity combined with the inherent wavelength independence of the nanomechanical sensor presents a competitive alternative to established tools for the analysis and localization of nonfluorescent single molecules and nanoparticles.

scholarly journals High-density super-resolution microscopy with an incoherent light source and a conventional epifluorescence microscope setup

2017 ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Light Source ◽  
Single Molecule ◽  
Super Resolution ◽  
High Density ◽  
Objective Lens ◽  
Incoherent Light ◽  
Superresolution Microscopy ◽  
Correlative Imaging ◽  
Deep Blue ◽  
Epifluorescence Microscope

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as Omnipresent Localisation Microscope (OLM), is an extension of Single Molecule Localisation Microscopy (SMLM) techniques and allows single molecules to be switched on and off (’blinking’), detected and localised. The use of a short burst of deep blue excitation can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, for the first time, we demonstrate that STED and OLM can be performed on the same biological sample using a simple imaging buffer. It is hoped that such a correlative imaging will provide a basis for a further enhanced resolution.Scope of the findingsDespite ten years of development, superresolution microscopy is still limited to relatively few microscopy and optics groups. This is mainly due to the significant cost of the superresolution microscopes which require high-quality lasers, high NA objective lens, a very sensitive camera, a highly precise microscope stage, and a complex post-acquisition data reconstruction and analysis. We present results that demonstrate the possibility to obtain nanoscale resolution images using a conventional microscope and an incoherent light source. We show an easyto-follow protocol that every biologist can implement in the laboratory. We hope that this finding will help any scientist to generate high-density super-resolution images even with limited budget. Lastly, the new photophysical observations reported here will pave the way for more in-depth investigations on excitation, photobleaching and photoactivation of a fluorophore.

scholarly journals Extracting microtubule networks from superresolution single-molecule localization microscopy data

2017 ◽  
Vol 28 (2) ◽  
pp. 333-345 ◽  
Author(s):  
Zhen Zhang ◽  
Yukako Nishimura ◽  
Pakorn Kanchanawong
Keyword(s):  
Quantitative Analysis ◽  
Single Molecule ◽  
Network Architecture ◽  
Spatial Organization ◽  
Data Sets ◽  
Cellular Functions ◽  
Microtubule Network ◽  
Superresolution Microscopy ◽  
Microtubule Networks ◽  
Filament Networks

Microtubule filaments form ubiquitous networks that specify spatial organization in cells. However, quantitative analysis of microtubule networks is hampered by their complex architecture, limiting insights into the interplay between their organization and cellular functions. Although superresolution microscopy has greatly facilitated high-resolution imaging of microtubule filaments, extraction of complete filament networks from such data sets is challenging. Here we describe a computational tool for automated retrieval of microtubule filaments from single-molecule-localization–based superresolution microscopy images. We present a user-friendly, graphically interfaced implementation and a quantitative analysis of microtubule network architecture phenotypes in fibroblasts.

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 Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

Interfacial charge transfer events of BODIPY molecules: single molecule spectroelectrochemistry and substrate effects

2014 ◽  
Vol 16 (42) ◽  
pp. 23150-23156 ◽  
Author(s):  
Jia Liu ◽  
Caleb M. Hill ◽  
Shanlin Pan ◽  
Haiying Liu
Keyword(s):  
Charge Transfer ◽  
Single Molecule ◽  
Single Molecules ◽  
Transfer Mechanism ◽  
Substrate Effects ◽  
Interfacial Charge Transfer ◽  
Charge Transfer Mechanism ◽  
Nanostructured Substrates ◽  
Interfacial Charge

BODIPY dye single molecules on nanostructured substrates are studied with a single molecule spectroelectrochemistry technique to reveal the heterogeneous charge transfer mechanism.

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