Combining confocal and single molecule localisation microscopy: A correlative approach to multi-scale tissue imaging

Methods ◽  
2015 ◽  
Vol 88 ◽  
pp. 98-108 ◽  
Author(s):  
David J. Crossman ◽  
Yufeng Hou ◽  
Isuru Jayasinghe ◽  
David Baddeley ◽  
Christian Soeller
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abdullah O. Khan ◽  
Carl W. White ◽  
Jeremy A. Pike ◽  
Jack Yule ◽  
Alexandre Slater ◽  
...  

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.


PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0125438 ◽  
Author(s):  
Matthieu Palayret ◽  
Helen Armes ◽  
Srinjan Basu ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  

2021 ◽  
Vol 7 (12) ◽  
pp. 266
Author(s):  
Bastien Laville ◽  
Laure Blanc-Féraud ◽  
Gilles Aubert

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.


2019 ◽  
Author(s):  
Ida S. Opstad ◽  
Florian Ströhl ◽  
Marcus Fantham ◽  
Colin Hockings ◽  
Oliver Vanderpoorten ◽  
...  

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.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Piotr Michaluk ◽  
Janosch Peter Heller ◽  
Dmitri A Rusakov

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.


2021 ◽  
Author(s):  
Jinmei Cheng ◽  
Edward S. Allgeyer ◽  
Jennifer H. Richens ◽  
Edo Dzafic ◽  
Amandine Palandri ◽  
...  

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.


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