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Quantification method

A short explanation of the method to quantify the actin response based on the skewness of the histogram is given here (unpublished material, therefore protected by password).

Quantitative Image Analysis

Cell biology is often perceived as the science of beautiful images. However, images contain much more information than usually exploited. Functional analysis is often based on comparisons (such as different concentrations of a compound, mutant versus wild type, different time points). How to do this with images? It is possible - when the visual information hidden in images is quantified. This is the idea of quantitative image analysis. Each application requires a specific approach, but there are some common principles that are helpful to avoid misinterpretations.

Quantitative image analysis is generally conducted by support of software, which is often very expensive (typically in the range of several 10 k€). However, there are versatile open-source alternatives such as the programme ImageJ, which allows to do most investigations free of charge, if one knows what one wants to do.

Your task will be to exercise this approach using a typical case study: in frame of a study on plant defence, we have discovered that a compound from the plant gan cao, used in Traditional Chinese Medicine, is not only active in the human body (by relaxing the smooth muscles in the bronchia, helping to soothe asthma), but also works with plant actin. This is then followed by induction of plant immunity. Using this discovery, Hao Wang, a Ph.D. student in the lab, investigated, whether gan cao could be used for biological plant protection (as immunostimulans) in vineyards to reduce fungicide application. In one experiment, he used transgenic grape plants that express a GFP-fusion of the actin-binding domain 2 of plant fimbrin, such that actin filaments can be observed by spinning disc microscopy in living plants. He tested now the effect of glycyrrhizine, the active compound from gan cao, on actin filaments. As a control, he used the solvent (water + 0.1% TWEEN) used to dissolve and apply glycyrrhizine. He conduted time course studies in leaf discs and was able to observe a drastic remodelling of actin in response to 2 mM glycyrrhizine, which was not seen in the control. Can you use those images to quantify the actin response?

  • Here you can download the programme and also get access to informational ressources, including plugins for specific applications: IMAGE J Webpage
  • Here you find some background on the principles of quantitative image analysis, since this is containing unpublished material, it is protected by password: unpublished material
  • Here you find the DATA SET (sample images in tiff-format from two experiments, a and b, control is solvent control, gly is treated with glycyrrhizine, each experiments has four time points, 0, 10 min, 15 min, and 30 min, zipped folder, right click and download)