Automating multimodal microscopy with NanoJ-Fluidics

Almada, Pedro; Pereira, Pedro M.; Culley, Siân; Caillol, Ghislaine; Boroni-Rueda, Fanny; Dix, Christina L.; Charras, Guillaume; Baum, Buzz; Laine, Romain F.; Leterrier, Christophe; Henriques, Ricardo

doi:10.1038/s41467-019-09231-9

Cited by 91 publications

(86 citation statements)

References 34 publications

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“…Neglecting to recognize this cost associated with increased resolution could render imaging results useless or worse might incorrectly inform researchers about a biological system. To demonstrate sample preparation inadequacies in imaging regimes, we took advantage of NanoJ-Fluidics (28) and NanoJ-SQUIRREL (21) to compare the pre-and post-fixation actin structures and CD4 cellular organization, in the same cells. We asked what would be the influence of chemical fixation using different imaging regimes with increasing resolution (TIRF, SIM and SMLM) by correlating pre-and post-fixation images.…”

Section: Suboptimal Fixation Protocols Affect the Actin Cytoskeleton mentioning

confidence: 99%

“…Hence, when performing essential protocol optimization, preservation of the overall cellular structure should be a priority. This work also aims to highlight that there are already established protocols that serve as excellent starting points (22,23,27), hardware that permits the optimization of such protocols to be streamlined (28,41) and tools that permit seamless analysis of possible bottlenecks (21,41). In conclusion, to extract the most from SMLM experiments it is essential to use reliable and repeatable imaging protocols that preserve, as much as possible, the overall cellular structure.…”

Section: Suboptimal Fixation Protocols Affect the Actin Cytoskeleton mentioning

confidence: 99%

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Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Pereira¹,

Albrecht²,

Jacobs³

et al. 2018

Preprint

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Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (∼ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy. Super-resolution microscopy | Single Molecule Localization Microscopy | NanoJ-Fluidics | NanoJ-SQUIRREL | Clustering analysis | Fixation | Artefacts | Actin cytoskeleton | CD4 membrane receptorCorrespondence: p.pereira@ucl.ac.uk, d.albrecht@ucl.ac.uk, r.henriques@ucl.ac.uk European Research Council (649101-UbiProPox) (J.M.); the MRC Programme Grant (MC_U12016/1) (M.M.); D.A. is presently a Marie Curie fellow (Marie Sklodowska-Curie grant agreement No 750673); C.J. was funded by a Commonwealth scholarship, funded by the UK government. AUTHOR CONTRIBUTIONS These contributions follow the Contributor Roles Taxonomy guidelines: . Conceptualization: P.M.P., D.A., C.J., R.H.; Data curation: P.M.P., D.A., C.J.; Formal analysis: P.M.P., D.A., C.J.; Investigation: P.M.P., D.A., C.J.; Methodology: P.M.P., D.A., C.J.; Resources: P.M.P., D.A., C.J., J.M., M.M., R.H.; Validation: P.M.P., D.A., C.J., J.M., M.M., R.H.; Visualization: P.M.P., D.A., C.J.; Writing original draft: P.M.P., D.A., C.J.; Writing, review & editing: P.M.P., D.A., C.J., J.M., M.M., R.H.; Funding acquisition: P.M.P., D.A., J.M., M.M., R.H.; Project administration: P.M.P., D.A., R.H.; Supervi-sion: P.M.P., D.A., J.M., M.M., R.H.

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Section: Suboptimal Fixation Protocols Affect the Actin Cytoskeleton mentioning

confidence: 99%

Section: Suboptimal Fixation Protocols Affect the Actin Cytoskeleton mentioning

confidence: 99%

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Pereira¹,

Albrecht²,

Jacobs³

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Moreover, fluorophores are constantly renewed at the sample as new imagers interact with the docking strands, allowing to accumulate a large number of localizations. Finally, multi-color DNA-PAINT is straightforward by using orthogonal docking strands on distinct secondary antibodies and corresponding imagers, allowing to image 5-8 different targets Almada et al, 2019;Yu Wang et al, 2017;Werbin et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2019

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Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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“…These aim for a careful trade-off between performance and cost, instead of maximizing system performance. This goal can be achieved by a combination of open-source / open-access software and hardware blueprints, as well as repurposing commodity industrial or consumer hardware [3][4][5][6][7][8][9] .…”

Section: Introductionmentioning

confidence: 99%

Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

Eynde

Sandmeyer

Vandenberg

et al. 2018

Preprint

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Super-Resolution (SR) fluorescence microscopy is typically carried out on high-end research microscopes. Super-resolution Optical Fluctuation Imaging (SOFI) is a fast SR technique capable of livecell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing super-resolution imaging techniques.Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFI-specific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

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Automating multimodal microscopy with NanoJ-Fluidics

Cited by 91 publications

References 34 publications

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Quantitative comparison of camera technologies for cost-effective Super-resolution Optical Fluctuation Imaging (SOFI)

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