Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy

Zhang, Ying; Huang, Tao; Jorgens, Danielle; Nickerson, Andrew; Lin, Li Jung; Pelz, Joshua; Gray, Joe W.; López, Clàudia; Nan, Xiaolin

doi:10.1371/journal.pone.0176839

Cited by 45 publications

(43 citation statements)

References 30 publications

(34 reference statements)

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“…Ideally, chemical fixation preserves the macroscopic structure of the sample as well as the native nanoscale organization of target proteins. However, true preservation at the subcellular level is not trivial, as known from electron microscopy studies (24). Furthermore, chemical fixation does not immediately immobilize membrane-associated proteins (25).…”

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confidence: 99%

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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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

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“…Processing of the corneal samples, including dissection of the rims into segments and chemical fixation, could potentially alter the relevant microanatomy. Cellular boundary retraction in response to SEM processing has been described previously, 45 and may result in underestimation of the cellular TZ width compared to the collagenous TM. However, with the resolution of current OCT images, the TZ borders cannot be directly identified in vivo.…”

Section: Discussionmentioning

confidence: 80%

Quantification of the Posterior Cornea Using Swept Source Optical Coherence Tomography

Wahlig

Yam

Chong

et al. 2018

Trans. Vis. Sci. Tech.

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PurposeWe define optical coherence tomography (OCT) measurement parameters of the corneal endothelium/Descemet's membrane (DM) complex and peripheral transition zone (TZ) and describe these measurements in an ethnically Chinese population.MethodsOCT images of the anterior segment and iridocorneal angle were obtained from 129 healthy Chinese subjects (129 eyes), aged 40 to 81 years. The scleral spur (SS) and Schwalbe's line (SL) were identified in each image. Endothelium/DM diameter, referred to as endothelial arc length (EAL), is the SL-to-SL distance. The SS-to-SL distance encompasses the TZ and trabecular meshwork (TM). Since the TZ cannot be visualized by OCT, a ratio of TZ-to-TZ+TM width was calculated from scanning electron microscopy (SEM) images obtained from 5 cadaveric corneas. The SS-to-SL distance was multiplied by this ratio to approximate in vivo TZ width.ResultsFrom SEM measurements, the relationship TZ = 0.20*(TZ+TM) was determined. From OCT measurements, mean EAL was 12.15 ± 0.58 mm and mean TZ width was 156 ± 20 μm. For eyes with horizontal and vertical images, vertical EAL was significantly greater than horizontal EAL (P = 0.03).ConclusionsCorneal endothelium/DM diameter and TZ width can be obtained from OCT images. Although only combined TZ+TM is visualized on OCT, TZ width can be reasonably approximated.Translational RelevanceEmerging procedures, like endothelial cell injection and DM transplantation (DMT), require accurate measurements of endothelium/DM size for preoperative planning. Size of the TZ, which may contain progenitor cells, also could contribute to endothelial regeneration in these procedures.

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“…For scanning electron microscopy (SEM), the cells were fixed overnight at 4 °C in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). A drop of sample was spreaded on a cover slip, air-dried, sputter-coated with colloidal gold and the morphology was examined under scanning electron microscope (EVO18 SEM, Zeiss) at an operating voltage of 15 kV 45,46 .…”

Section: Effect Of Flavonoids On the Cell Wall Damagementioning

confidence: 99%

Screening of natural compounds that targets glutamate racemase of Mycobacterium tuberculosis reveals the anti-tubercular potential of flavonoids

Pawar

Jha

Chopra

et al. 2020

Sci Rep

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tuberculosis (tB) is caused by Mycobacterium tuberculosis (MTB), a highly infectious disease accounting for nearly 1.5 million deaths every year and has been a major global concern. Moreover, resistance to anti-TB drugs is an arduous obstacle to effective prevention, TB care and management. Therefore, incessant attempts are being made to identify novel drug targets and newer anti-tubercular drugs to fight with this deadly pathogen. Increasing resistance, adverse effects and costly treatment by conventional therapeutic agents have been inclining the researchers to search for an alternative source of medicine. In this regard natural compounds have been exploited extensively for their therapeutic interventions targeting cellular machinery of MTB. Glutamate racemase (MurI) is an enzyme involved in peptidoglycan (PG) biosynthesis and has become an attractive target due to its moonlighting property. We screened various classes of natural compounds using computational approach for their binding to MTB-MurI. Shortlisted best docked compounds were evaluated for their functional, structural and anti-mycobacterial activity. The results showed that two flavonoids (naringenin and quercetin) exhibited best binding affinity with MTB-MurI and inhibited the racemization activity with induced structural perturbation. In addition, fluorescence and electron microscopy were employed to confirm the membrane and cell wall damages in mycobacterial cells on exposure to flavonoids. Together, these observations could provide impetus for further research in better understanding of anti-tubercular mechanisms of flavonoids and establishing them as lead molecules for TB treatment.

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Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy

Cited by 45 publications

References 30 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

Quantification of the Posterior Cornea Using Swept Source Optical Coherence Tomography

Screening of natural compounds that targets glutamate racemase of Mycobacterium tuberculosis reveals the anti-tubercular potential of flavonoids

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