Dataset Information

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

ABSTRACT: We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1? on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications.

SUBMITTER: Mayr S

PROVIDER: S-EPMC7384682 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

Mayr Sandra S Hauser Fabian F Puthukodan Sujitha S Axmann Markus M Göhring Janett J Jacak Jaroslaw J

PLoS computational biology 20200630 6

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. ...[more]

PMID: 32603371

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Project description:BACKGROUND: According to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called 'minimum required performance limit' (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable. RESULTS: We developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section 'Availability of supporting data' for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less. CONCLUSIONS: The aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in -but not limited to- the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Dataset Information

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

Publications

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

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