Designing a rigorous microscopy experiment: Validating methods and avoiding bias

Jost, Anna Payne-Tobin; Waters, Jennifer

doi:10.1083/jcb.201812109

Cited by 85 publications

(77 citation statements)

References 103 publications

(173 reference statements)

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“…We do not have a good explanation for this, but it is worth noting that while formal training in biochemistry, genetics, and molecular and cellular biology is mandatory in most undergraduate and graduate biomedical programs, microscopy and imaging are rarely part of the curriculum. Our suspicion is that authors are not quite sure as to what really matters in an imaging experiment ( Jost and Waters, 2019 ; North, 2006 ). It is interesting to note that the Nature Research Life Sciences Reporting Summary includes specific and detailed questionnaires for antibodies, cell lines, statistical analysis, ChIP-Seq, flow cytometry, and MRI, but not for optical imaging ( https://www.nature.com/documents/nr-reporting-summary-flat.pdf ).…”

Section: Discussionmentioning

confidence: 99%

Imaging methods are vastly underreported in biomedical research

Marqués

Pengo

Sanders

2020

eLife

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A variety of microscopy techniques are used by researchers in the life and biomedical sciences. As these techniques become more powerful and more complex, it is vital that scientific articles containing images obtained with advanced microscopes include full details about how each image was obtained. To explore the reporting of such details we examined 240 original research articles published in eight journals. We found that the quality of reporting was poor, with some articles containing no information about how images were obtained, and many articles lacking important basic details. Efforts by researchers, funding agencies, journals, equipment manufacturers and staff at shared imaging facilities are required to improve the reporting of experiments that rely on microscopy techniques.

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Section: Discussionmentioning

confidence: 99%

Imaging methods are vastly underreported in biomedical research

Marqués

Pengo

Sanders

2020

eLife

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“…When using a microscopy image, it is important to remember that we are looking at an image and not the biological specimen. Further we must consider sources of errors in the process and if the image truly represents the biology of the specimen (159). For a long time, one of the major limitations of microscopy has been the diffraction limit.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

“…Equally important is to choose the correct approach and technique for the question in mind. It is important to use known samples and control experiments to validate methods and identify errors, and test approaches for error correction (159). In this thesis we have used wide variety of microscopy methods (Figure 16), each of these methods has their advantages and disadvantages.…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Class III phosphatidylinositol-3-OH kinase controls epithelial integrity through endosomal LKB1 regulation

et al. 2017

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The molecular mechanisms underlying the interdependence between intracellular trafficking and epithelial cell polarity are poorly understood. Here we show that inactivation of class III phosphatidylinositol-3-OH kinase (CIII-PI3K), which produces phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes, disrupts epithelial organization. This is caused by dysregulation of endosomally localized Liver Kinase B1 (LKB1, also known as STK11), which shows delocalized and increased activity accompanied by dysplasia-like growth and invasive behaviour of cells provoked by JNK pathway activation. CIII-PI3K inactivation cooperates with Ras to promote tumour growth in vivo in an LKB1-dependent manner. Strikingly, co-depletion of LKB1 reverts these phenotypes and restores epithelial integrity. The endosomal, but not autophagic, function of CIII-PI3K controls polarity. We identify the CIII-PI3K effector, WD repeat and FYVE domain-containing 2 (WDFY2), as an LKB1 regulator in Drosophila tissues and human organoids. Thus, we define a CIII-PI3K-regulated endosomal signalling platform from which LKB1 directs epithelial polarity, the dysregulation of which endows LKB1 with tumour-promoting properties.

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“…One of the greatest barriers to rigorous and reproducible confocal microscope experiments is encountered before an image is even taken: how do you select the field of view in an unbiased way ? Unlike other disciplines where blinded experiments are routine, confocal microscopy is often performed by the same person who prepares the specimens.…”

Section: Selection Biasmentioning

confidence: 99%