Tutorial: guidance for quantitative confocal microscopy

Jonkman, James; Brown, Claire M.; Wright, Graham D.; Anderson, Kurt I.; North, Alison J.

doi:10.1038/s41596-020-0313-9

Cited by 273 publications

(215 citation statements)

References 92 publications

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“…Although the increase in immunostaining demonstrated for FN ED-A and FN ED-B in GIMs is qualitative in the current study, quantitative tools in confocal microscopy in addition to multiple redundant approaches may be essential to definitively document the precise relative quantities of these isoforms in future investigation. 102,103…”

Section: Discussionmentioning

confidence: 99%

Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

Yemanyi

Baidouri

Burns

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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Section: Discussionmentioning

confidence: 99%

Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

Yemanyi

Baidouri

Burns

et al. 2020

Invest. Ophthalmol. Vis. Sci.

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“…The result of statistical analysis is listed in the form of a table of all classes of data from 3 different experiments. The standard procedure for the statistical analysis can be found elsewhere [47]. The detailed information about all 3 experiments and their analysis is provided in appendix A.…”

Section: Sim Imaging Of the Cell Membrane And Mitochondriamentioning

confidence: 99%

“…It is important to repeat experiment several times to ensure that the results are reproducible. We have performed statistical analysis on data from 3 different experiments and due to inherent biological variation, it is expected that variability between the mean of experimental data is high in some cases [47,50]. The results for macrophages and trophoblast are shown in Figs.…”

Section: Appendix A: Statistical Assessment Of Sim Images For Experimmentioning

confidence: 99%

Quantitative assessment of morphology and sub-cellular changes in macrophages and trophoblasts during inflammation

Singh¹,

Dubey²,

Wolfson³

et al. 2020

Biomed. Opt. Express

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In pregnancy during an inflammatory condition, macrophages present at the fetomaternal junction release an increased amount of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α and INF-γ, which can disturb the trophoblast functions and pregnancy outcome. Measurement of the cellular and sub-cellular morphological modifications associated with inflammatory responses are important in order to quantify the extent of trophoblast dysfunction for clinical implication. With this motivation, we investigated morphological, cellular and sub-cellular changes in externally inflamed RAW264.7 (macrophage) and HTR-8/SVneo (trophoblast) using structured illumination microscopy (SIM) and quantitative phase microscopy (QPM). We monitored the production of NO, changes in cell membrane and mitochondrial structure of macrophages and trophoblasts when exposed to different concentrations of proinflammatory agents (LPS and TNF-α). In vitro NO production by LPS-induced macrophages increased 22-fold as compared to controls, whereas no significant NO production was seen after the TNF-α challenge. Under similar conditions as with macrophages, trophoblasts did not produce NO following either LPS or the TNF-α challenge. Super-resolution SIM imaging showed changes in the morphology of mitochondria and the plasma membrane in macrophages following the LPS challenge and in trophoblasts following the TNF-α challenge. Label-free QPM showed a decrease in the optical thickness of the LPS-challenged macrophages while TNF-α having no effect. The vice-versa is observed for the trophoblasts. We further exploited machine learning approaches on a QPM dataset to detect and to classify the inflammation with an accuracy of 99.9% for LPS-challenged macrophages and 98.3% for TNF-α-challenged trophoblasts. We believe that the multi-modal advanced microscopy methodologies coupled with machine learning approach could be a potential way for early detection of inflammation.

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“…Fluorescence microscopy analysis (namely analysis of lysosomal size and circularity, colocalization of proteins Rab7/LAMP1, cellular localization of p53) was subjected to quantitative assessment in Cells 2020, 9, 1015 6 of 24 accordance with rigorously defined guidelines [47]. For a quantitative analysis of the images, we utilized the published guidance for quantitative confocal microscopy [48,49]. Images from three independent experiments were subjected to quantitative analysis.…”

Section: Discussionmentioning

confidence: 99%

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

Uzhytchak

Smolková

Lunová

et al. 2020

Cells

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Iron oxide-based nanoparticles have been repeatedly shown to affect lysosomal-mediated signaling. Recently, nanoparticles have demonstrated an ability to modulate autophagic flux via lysosome-dependent signaling. However, the precise underlying mechanisms of such modulation as well as the impact of cellular genetic background remain enigmatic. In this study, we investigated how lysosomal-mediated signaling is affected by iron oxide nanoparticle uptake in three distinct hepatic cell lines. We found that nanoparticle-induced lysosomal dysfunction alters sub-cellular localization of pmTOR and p53 proteins. Our data indicate that alterations in the sub-cellular localization of p53 protein induced by nanoparticle greatly affect the autophagic flux. We found that cells with high levels of Bcl-2 are insensitive to autophagy initiated by nanoparticles. Altogether, our data identify lysosomes as a central hub that control nanoparticle-mediated responses in hepatic cells. Our results provide an important fundamental background for the future development of targeted nanoparticle-based therapies.

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Tutorial: guidance for quantitative confocal microscopy

Cited by 273 publications

References 92 publications

Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

Quantitative assessment of morphology and sub-cellular changes in macrophages and trophoblasts during inflammation

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

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