scholarly journals Single-Molecule Clustering for Super-Resolution Optical Fluorescence Microscopy

Photonics ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 7
Author(s):  
Prakash Joshi ◽  
Partha Pratim Mondal
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Physiological State ◽  
Super Resolution ◽  
Molecular Clusters ◽  
Pairwise Distance ◽  
Critical Parameters ◽  
Clustering Methods ◽  
Biophysical Parameters ◽  
Nih3t3 Cells

Molecular assembly in a complex cellular environment is vital for understanding underlying biological mechanisms. Biophysical parameters (such as single-molecule cluster density, cluster-area, pairwise distance, and number of molecules per cluster) related to molecular clusters directly associate with the physiological state (healthy/diseased) of a cell. Using super-resolution imaging along with powerful clustering methods (K-means, Gaussian mixture, and point clustering), we estimated these critical biophysical parameters associated with dense and sparse molecular clusters. We investigated Hemaglutinin (HA) molecules in an Influenza type A disease model. Subsequently, clustering parameters were estimated for transfected NIH3T3 cells. Investigations on test sample (randomly generated clusters) and NIH3T3 cells (expressing Dendra2-Hemaglutinin (Dendra2-HA) photoactivable molecules) show a significant disparity among the existing clustering techniques. It is observed that a single method is inadequate for estimating all relevant biophysical parameters accurately. Thus, a multimodel approach is necessary in order to characterize molecular clusters and determine critical parameters. The proposed study involving optical system development, photoactivable sample synthesis, and advanced clustering methods may facilitate a better understanding of single molecular clusters. Potential applications are in the emerging field of cell biology, biophysics, and fluorescence imaging.

scholarly journals Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Super-Resolution Image Reconstruction Based on Single-Molecule Localization Algorithm

Photonics ◽  
2021 ◽  
Vol 8 (7) ◽  
pp. 273
Author(s):  
Lixin Liu ◽  
Meijie Qi ◽  
Yujie Liu ◽  
Xinzhu Xue ◽  
Danni Chen ◽  
...  
Keyword(s):  
Image Reconstruction ◽  
Single Molecule ◽  
Cell Biology ◽  
Imaging System ◽  
Particle Density ◽  
Super Resolution ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Localization Algorithm ◽  
Fluorescent Molecules

Fluorescence imaging is an important and efficient tool in cell biology and biomedical research. In order to observe the dynamics of biological macromolecules such as DNA, RNA and proteins in live cells, it is extremely necessary to surpass the Abbe diffraction limit in microscopic imaging. Single-molecule localization microscopy (SMLM) is a sort of super-resolution imaging technique that can obtain a large number of images of sparse fluorescent molecules by the use of photoswitchable fluorescent probes and single-molecule localization technology. The center positions of fluorescent molecules in the images are precisely located, and then the entire sample pattern is reconstructed with super resolution. In this paper, we present a single-molecule localization algorithm (SMLA) that is based on blind deconvolution and centroid localization (BDCL) method. Single-molecule localization and image reconstruction of 15,000/9990 frames of original images of tubulins are accomplished. In addition, this fluorophore localization algorithm is used to localize high particle-density images. The results show that our BDCL-SMLA method is a reasonable attempt and useful method for SMLM imaging when the imaging system is unknown.

scholarly journals Comparing Lifeact and Phalloidin for Super-resolution Imaging of Actin in Fixed Cells

2020 ◽  
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

AbstractVisualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

2020 ◽  
Author(s):  
Partha Pratim Mondal
Keyword(s):  
Distribution Function ◽  
Single Molecule ◽  
Gaussian Function ◽  
Single Molecules ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
Image Resolution ◽  
Size Spectrum ◽  
Nih3t3 Cells ◽  
Microscopy Technique

To be able to resolve molecular-clusters it is crucial to access vital informations (such as, molecule density and cluster-size) that are key to understand disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precisions (LPs)) to reconstruct super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single molecules) that degrade image resolution. Ideally it should be possible to identify the brightest molecules (also termed as, the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. POSSIBLE microscopy explores this possibility by introducing narrow probability size-distribution of single molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single molecule filtering is carried out by the Gaussian distribution function to filter out unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques.

scholarly journals A correlation analysis framework for localization-based super-resolution microscopy

2017 ◽  
Author(s):  
Joerg Schnitzbauer ◽  
Yina Wang ◽  
Matthew Bakalar ◽  
Baohui Chen ◽  
Tulip Nuwal ◽  
...  
Keyword(s):  
Correlation Analysis ◽  
Single Molecule ◽  
Quantitative Description ◽  
Super Resolution ◽  
Analysis Framework ◽  
Biophysical Parameters ◽  
Wide Range ◽  
The Time Domain ◽  
Analysis Platform ◽  
Super Resolution Microscopy

AbstractSuper-resolution images reconstructed from single-molecule localizations can reveal cellular structures close to the macromolecular scale and are now being used routinely in many biomedical research applications. However, because of their coordinate-based representation, a widely applicable and unified analysis platform that can extract a quantitative description and biophysical parameters from these images is yet to be established. Here, we propose a conceptual framework for correlation analysis of coordinate-based super-resolution images using distance histograms. We demonstrate the application of this concept in multiple scenarios including image alignment, tracking of diffusing molecules, as well as for quantification of colocalization.Significance statementCorrelation analysis is one of the most widely used image processing method. In the quantitative analysis of localization-based super-resolution images, there still lacks a generalized coordinate-based correlation analysis framework to take fully advantage of the super-resolution information. We show a coordinate-based correlation analysis framework for localization-based super-resolution microscopy. This framework is highly general and flexible in that it can be easily extended to model the effect of localization uncertainty, to the time domain and other distance definitions, enabling it to be adapted for a wide range of applications. Our work will greatly benefit the quantitative interpretation of super-resolution images and thus the biological application of super-resolution microscopy.

scholarly journals Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

2021 ◽  
Author(s):  
Kaarjel K. Narayanasamy ◽  
Aleksandar Stojic ◽  
Yunqing Li ◽  
Steffen Sass ◽  
Marina Hesse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Super Resolution ◽  
Protein Patterns ◽  
Multiple Targets ◽  
Localization Microscopy ◽  
Neuronal Tissue ◽  
Tissue Sections ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractThe development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

scholarly journals A Pairwise Distance Distribution Correction (DDC) algorithm to eliminate blinking-caused artifacts in super-resolution microscopy

2019 ◽  
Author(s):  
Christopher H. Bohrer ◽  
Xinxing Yang ◽  
Xiaoli Weng ◽  
Brian Tenner ◽  
Shreyasi Thakur ◽  
...  
Keyword(s):  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Distance Distribution ◽  
Pairwise Distance ◽  
Imaging Sequence ◽  
Wide Range ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Accurate Reconstruction

AbstractIn single-molecule localization based super-resolution microscopy (SMLM), a fluorophore stochastically switches between fluorescent- and dark-states, leading to intermittent emission of fluorescence, a phenomenon known as blinking. Intermittent emissions create multiple localizations belonging to the same molecule, resulting in blinking-artifacts within SMLM images. These artifacts are often interpreted as true biological assemblies, confounding quantitative analyses and interpretations. Multiple methods have been developed to eliminate these artifacts, but they either require additional experiments, arbitrary thresholds, or specific photo-kinetic models. Here we present a method, termed Distance Distribution Correction (DDC), to eliminate blinking-caused repeat localizations without any additional calibrations. The approach relies on the finding that the true pairwise distance distribution of different fluorophores in an SMLM image can be naturally obtained from the imaging sequence by using distances between localizations separated by a time much longer than the average fluorescence survival time. We show that using the true pairwise distribution we can define and then maximize the likelihood of obtaining a particular set of localizations void of blinking-artifacts, generating an accurate reconstruction of the underlying cellular structure. Using both simulated and experimental data, we show that DDC surpasses all previous existing blinking-artifact correction methodologies, resulting in drastic improvements in obtaining the closest estimate of the true spatial organization and number of fluorescent emitters in a wide range of applications. The simplicity and robustness of DDC will allow it to become the field standard in SMLM imaging, enabling the most accurate reconstruction and quantification of SMLM images to date.

scholarly journals Nanocarriers used as probes for super-resolution microscopy

2021 ◽  
Author(s):  
Sreejesh Sreedharan ◽  
Rajeshwari Tiwari ◽  
Deepak Tyde ◽  
Stephen O. Aderinto ◽  
Sumit Kumar Pramanik ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Super Resolution ◽  
Cellular Structures ◽  
Super Resolution Microscopy ◽  
Resolution Single

Super-resolution microscopy (SRM) has revolutionized cell biology, enabling visualization of cellular structures with nanometric resolution, single-molecule sensitivity, and with multiple colors. Here we review how nanocontainers have been used to enhance these techniques.

scholarly journals New ways of looking at very small holes – using optical nanoscopy to visualize liver sinusoidal endothelial cell fenestrations

Nanophotonics ◽  
2018 ◽  
Vol 7 (3) ◽  
pp. 575-596 ◽  
Author(s):  
Cristina I. Øie ◽  
Viola Mönkemöller ◽  
Wolfgang Hübner ◽  
Mark Schüttpelz ◽  
Hong Mao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Super Resolution ◽  
Living Cells ◽  
Liver Sinusoidal Endothelial Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Liver Sinusoidal Endothelial Cells ◽  
Dynamic Structures

AbstractSuper-resolution fluorescence microscopy, also known as nanoscopy, has provided us with a glimpse of future impacts on cell biology. Far-field optical nanoscopy allows, for the first time, the study of sub-cellular nanoscale biological structures in living cells, which in the past was limited to electron microscopy (EM) (in fixed/dehydrated) cells or tissues. Nanoscopy has particular utility in the study of “fenestrations” – phospholipid transmembrane nanopores of 50–150 nm in diameter through liver sinusoidal endothelial cells (LSECs) that facilitate the passage of plasma, but (usually) not blood cells, to and from the surrounding hepatocytes. Previously, these fenestrations were only discernible with EM, but now they can be visualized in fixed and living cells using structured illumination microscopy (SIM) and in fixed cells using single molecule localization microscopy (SMLM) techniques such as direct stochastic optical reconstruction microscopy. Importantly, both methods use wet samples, avoiding dehydration artifacts. The use of nanoscopy can be extended to the in vitro study of fenestration dynamics, to address questions such as the following: are they actually dynamic structures, and how do they respond to endogenous and exogenous agents? A logical further extension of these methodologies to liver research (including the liver endothelium) will be their application to liver tissue sections from animal models with different pathological manifestations and ultimately to patient biopsies. This review will cover the current state of the art of the use of nanoscopy in the study of liver endothelium and the liver in general. Potential future applications in cell biology and the clinical implications will be discussed.

Sign in / Sign up

Export Citation Format

Share Document