scholarly journals A method for single molecule localization microscopy of tissues reveals nonrandom distribution of nuclear pores in Drosophila

2021 ◽  
Author(s):  
Jinmei Cheng ◽  
Edward S Allgeyer ◽  
Jennifer H Richens ◽  
Edo Dzafic ◽  
Amandine Palandri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Optical Sectioning ◽  
Nuclear Pores ◽  
High Background ◽  
Chromatin Organisation ◽  
Localisation Microscopy ◽  
Single Molecule Localization Microscopy

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30nm resolution or better at depths greater than 20 μm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate chromatin organisation locally and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

scholarly journals A single molecule localization microscopy method for tissues reveals nonrandom nuclear pore distribution in Drosophila

2021 ◽  
Author(s):  
Jinmei Cheng ◽  
Edward S. Allgeyer ◽  
Jennifer H. Richens ◽  
Edo Dzafic ◽  
Amandine Palandri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Nuclear Pore ◽  
Optical Sectioning ◽  
Nuclear Pores ◽  
High Background ◽  
Chromatin Organisation ◽  
Microscopy Method ◽  
Localisation Microscopy

Single Molecule Localisation Microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues, because of high background from out of focus emitters and optical aberrations. Here we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-PAINT routinely gives 30 nm resolution or better at depths greater than 20 µm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to cultured cells. Lamin Dm0 shows a complementary localisation to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C distributes the nuclear pores more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Since nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organisation and contribute to the disease phenotypes caused by human Lamin A/C laminopathies.

scholarly journals Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0125438 ◽  
Author(s):  
Matthieu Palayret ◽  
Helen Armes ◽  
Srinjan Basu ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Optical Sectioning ◽  
Resolution Imaging ◽  
Localisation Microscopy

scholarly journals FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

2020 ◽  
Vol 16 (12) ◽  
pp. e1008479
Author(s):  
Daniel F. Nino ◽  
Daniel Djayakarsana ◽  
Joshua N. Milstein
Keyword(s):  
Single Molecule ◽  
Clustering Algorithm ◽  
Spatial Clustering ◽  
Cluster Complex ◽  
Nuclear Pore ◽  
Data Sets ◽  
Localization Microscopy ◽  
Biological Dataset ◽  
Open Source Software Package ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) is a powerful tool for studying intracellular structure and macromolecular organization at the nanoscale. The increasingly massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Here we present FOCAL3D, an accurate, flexible and exceedingly fast (scaling linearly with the number of localizations) density-based algorithm for quantifying spatial clustering in large 3D SMLM data sets. Unlike DBSCAN, which is perhaps the most commonly employed density-based clustering algorithm, an optimum set of parameters for FOCAL3D may be objectively determined. We initially validate the performance of FOCAL3D on simulated datasets at varying noise levels and for a range of cluster sizes. These simulated datasets are used to illustrate the parametric insensitivity of the algorithm, in contrast to DBSCAN, and clustering metrics such as the F1 and Silhouette score indicate that FOCAL3D is highly accurate, even in the presence of significant background noise and mixed populations of variable sized clusters, once optimized. We then apply FOCAL3D to 3D astigmatic dSTORM images of the nuclear pore complex (NPC) in human osteosaracoma cells, illustrating both the validity of the parameter optimization and the ability of the algorithm to accurately cluster complex, heterogeneous 3D clusters in a biological dataset. FOCAL3D is provided as an open source software package written in Python.

scholarly journals Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Super Resolution ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Laser Illumination ◽  
Quantitative Analyses ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractNon-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies.

scholarly journals FOCAL3D: A 3-dimensional clustering package for single-molecule localization microscopy

2019 ◽  
Author(s):  
D. Nino ◽  
D. Djayakarsana ◽  
J. N. Milstein
Keyword(s):  
Single Molecule ◽  
Software Package ◽  
Clustering Algorithm ◽  
Image Resolution ◽  
Nuclear Pore ◽  
Localization Microscopy ◽  
3 Dimensional ◽  
Highly Efficient ◽  
Human Osteosarcoma Cells ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) yields an image resolution 1-2 orders of magnitude below that of conventional light microscopy, resolving fine details on intracellular structure and macromolecular organization. The massive pointillistic data sets generated by SMLM require the development of new and highly efficient quantification tools. Density based clustering algorithms, such as DBSCAN, can provide spatial statistics on protein/nucleic acid aggregation or dispersion while explicitly identifying macromolecular clusters. The performance of DBSCAN, however, is typically dependent upon an arbitrary, or at least highly subjective, parametric tuning of the algorithm. Moreover, DBSCAN can be computationally expensive, which makes it arduous to evaluate on large image stacks. This is all the more important in 3-dimensions where there exist limited alternatives for quantifying clustering in SMLM data, and where a 2-dimensional analysis of true 3-dimensional data may give rise to image artefacts. We have developed an open-source software package in Python for both identifying and quantifying spatial clustering in 3-dimensional SMLM datasets. FOCAL3D is an extension of our previously developed, 2-dimensional, grid based clustering algorithm FOCAL. FOCAL3D provides a highly efficient way to spatially cluster SMLM datasets, scaling linearly with the number of localizations, and the algorithmic parameters may be systematically optimized so that the resulting analysis is insensitive to variation over a range of parameter choices. We initially validate the performance and parametric insensitivity of FOCAL3D on simulated datasets, then apply the algorithm to 3-dimensional, astigmatic dSTORM images of the nuclear pore complex in human osteosarcoma cells.The data and software package are available at: http://www.utm.utoronto.ca/milsteinlab/software/

scholarly journals Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

2020 ◽  
Author(s):  
Adrien Mau ◽  
Karoline Friedl ◽  
Christophe Leterrier ◽  
Nicolas Bourg ◽  
Sandrine Lévêque-Fort
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Field Of View ◽  
Uniform Illumination ◽  
Optical Sectioning ◽  
Coated Pits ◽  
Uniform Size ◽  
Cos Cells ◽  
Quantitative Analyses ◽  
Single Molecule Localization Microscopy

AbstractQuantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussian-shaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical. This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2-spectrin along the axons of neurons. ASTER’s sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.

scholarly journals 3D particle averaging and detection of macromolecular symmetry in localization microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hamidreza Heydarian ◽  
Maarten Joosten ◽  
Adrian Przybylski ◽  
Florian Schueder ◽  
Ralf Jungmann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Fluorescent Labeling ◽  
Dna Origami ◽  
Particle Analysis ◽  
Nuclear Pore ◽  
Localization Microscopy ◽  
Structural Template ◽  
Microscopy Techniques ◽  
Single Molecule Localization Microscopy

AbstractSingle molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.

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