Investigating Chromatin Organisation Using Single Molecule Localisation Microscopy

2017 ◽  
pp. 25-61 ◽  
Author(s):  
Kirti Prakash
Keyword(s):  
Single Molecule ◽  
Chromatin Organisation ◽  
Localisation Microscopy

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 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 Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated knock-in

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abdullah O. Khan ◽  
Carl W. White ◽  
Jeremy A. Pike ◽  
Jack Yule ◽  
Alexandre Slater ◽  
...  
Keyword(s):  
Genome Editing ◽  
G Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Single Molecule Imaging ◽  
Dependent Effect ◽  
Systematic Comparison ◽  
Selection Strategies ◽  
Localisation Microscopy ◽  
G Protein Coupled

Abstract The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR-knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

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 Off-The-Grid Variational Sparse Spike Recovery: Methods and Algorithms

2021 ◽  
Vol 7 (12) ◽  
pp. 266
Author(s):  
Bastien Laville ◽  
Laure Blanc-Féraud ◽  
Gilles Aubert
Keyword(s):  
Inverse Problem ◽  
Single Molecule ◽  
Super Resolution ◽  
Research Field ◽  
Fine Scale ◽  
Seminal Work ◽  
Resolution Problem ◽  
Localisation Microscopy ◽  
New Research ◽  
Recovery Methods

Gridless sparse spike reconstruction is a rather new research field with significant results for the super-resolution problem, where we want to retrieve fine-scale details from a noisy and filtered acquisition. To tackle this problem, we are interested in optimisation under some prior, typically the sparsity i.e., the source is composed of spikes. Following the seminal work on the generalised LASSO for measures called the Beurling-Lasso (BLASSO), we will give a review on the chief theoretical and numerical breakthrough of the off-the-grid inverse problem, as we illustrate its usefulness to the super-resolution problem in Single Molecule Localisation Microscopy (SMLM) through new reconstruction metrics and tests on synthetic and real SMLM data we performed for this review.

scholarly journals A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view

2019 ◽  
Author(s):  
Ida S. Opstad ◽  
Florian Ströhl ◽  
Marcus Fantham ◽  
Colin Hockings ◽  
Oliver Vanderpoorten ◽  
...  
Keyword(s):  
Single Molecule ◽  
Hippocampal Neurons ◽  
Total Internal Reflection ◽  
Ganglion Cells ◽  
Cell Types ◽  
Live Cell ◽  
Retinal Ganglion ◽  
Photonic Waveguides ◽  
Set Up ◽  
Localisation Microscopy

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2] and have also been demonstrated for short-term live-imaging of robust cell types [3–5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGC). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

scholarly journals Rapid recycling of glutamate transporters on the astroglial surface

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Piotr Michaluk ◽  
Janosch Peter Heller ◽  
Dmitri A Rusakov
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Lateral Diffusion ◽  
Synaptic Cleft ◽  
Surface Pattern ◽  
Membrane Turnover ◽  
C Terminus ◽  
Localisation Microscopy

Glutamate uptake by astroglial transporters confines excitatory transmission to the synaptic cleft. The efficiency of this mechanism depends on the transporter dynamics in the astrocyte membrane, which remains poorly understood. Here, we visualise the main glial glutamate transporter GLT1 by generating its pH-sensitive fluorescent analogue, GLT1-SEP. FRAP-based imaging shows that 70-75% of GLT1-SEP dwell on the surface of rat brain astroglia, recycling with a lifetime of ~22 s. Genetic deletion of the C-terminus accelerates GLT1-SEP membrane turnover while disrupting its surface pattern, as revealed by single-molecule localisation microscopy. Excitatory activity boosts surface mobility of GLT1-SEP, involving its C-terminus, metabotropic glutamate receptors, intracellular Ca2+ and calcineurin-phosphatase activity, but not the broad-range kinase activity. The results suggest that membrane turnover, rather than lateral diffusion, is the main 'redeployment' route for the immobile fraction (20-30%) of surface-expressed GLT1. This finding reveals an important mechanism helping to control extrasynaptic escape of glutamate.

scholarly journals K-Neighbourhood Analysis: A Method for Understanding SMLM Images as Compositions of Local Neighbourhoods

2021 ◽  
Vol 1 ◽  
Author(s):  
Kristen Feher ◽  
Matthew S. Graus ◽  
Simao Coelho ◽  
Megan V. Farrell ◽  
Jesse Goyette ◽  
...  
Keyword(s):  
Single Molecule ◽  
Principal Component ◽  
Spatial Arrangement ◽  
Point Pattern ◽  
Analysis Tool ◽  
Nearest Neighbour ◽  
Clustering Methods ◽  
Neighbourhood Analysis ◽  
Localisation Microscopy ◽  
Novel Algorithm

Single molecule localisation microscopy (SMLM) is a powerful tool that has revealed the spatial arrangement of cell surface signalling proteins, producing data of enormous complexity. The complexity is partly driven by the convolution of technical and biological signal components, and partly by the challenge of pooling information across many distinct cells. To address these two particular challenges, we have devised a novel algorithm called K-neighbourhood analysis (KNA), which emphasises the fact that each image can also be viewed as a composition of local neighbourhoods. KNA is based on a novel transformation, spatial neighbourhood principal component analysis (SNPCA), which is defined by the PCA of the normalised K-nearest neighbour vectors of a spatially random point pattern. Here, we use KNA to define a novel visualisation of individual images, to compare within and between groups of images and to investigate the preferential patterns of phosphorylation. This methodology is also highly flexible and can be used to augment existing clustering methods by providing clustering diagnostics as well as revealing substructure within microclusters. In summary, we have presented a highly flexible analysis tool that presents new conceptual possibilities in the analysis of SMLM images.

scholarly journals Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

2021 ◽  
Author(s):  
Ruta Gerasimaite ◽  
Jonas Bucevicius ◽  
Kamila A. Kiszka ◽  
Georgij Kostiuk ◽  
Tanja Koenen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Single Molecule ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Linker Length ◽  
Localisation Microscopy ◽  
Red Dye ◽  
Small Molecule Probe

Here we report a small molecule probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of <50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.

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