(B) Densitometric analysis of AIF in the mitochondrial and nuclear protein. In the end, the integrity and robustness of the published scientific data should be part of the considerations while planning image analysis experiments. 1E was conducted using ImageJ oneway ANOVA was used for statistical analysis. Sometimes, a qualitative interpretation might be better suitable than a flawed quantification. IMHO: Not because we can measure everything means also we should do it. There are a number of different ways to get intensity information from images using the base package of ImageJ (no plugins required). ImageJ is useful for getting information from images, including pixel intensity. I can only recommend to use either the software of the Western Blot imaging system in the lab or alternatively GelAnalyzer because it allows to stick pretty well to the recommended procedure by Western Blot material suppliers (which I would guess are the most experienced people in that particular field) and as seen in the publication above. Basic Intensity Quantification with ImageJ Pretty pictures are nice, but many times we need to turn our images into quantifiable data. Hammond, “A defined methodology for reliable quantification of Western blot data.,” Mol. So, generally there are many pitfalls related to WB measurements.īesides the literature list in one of the linked posts above, mainly the following gives a good insight into the procedure: The lanes were always positioned at the center of the gel lane. The size of the lane selection tool was 16 pixels wide (Figure 2a), equivalent to 30 of the total width of the well as suggested by Gassmann et al. Anyway, in order to quantify the bands you need to get histogram of bands from each lane separately. The Gel Analyzer tool of ImageJ was used to determine the profiles of each lane of the gels. And band selections which overshoot the actual band also lead to wrong results. However, the mol wt of the bands are different from the standards. One cannot reliably quantify bands by making boxes around them and using analyze measure nor do horizontal lines fulfill the requirements. Here is one video which is explaining a little more detailed the considerations of WB in general, normalization as well as measurements The latter and pretty much of most other ones completely ignore every thing mentioned in scientific literature regarding Western Blot measurements (and the pre-requisites for it). There are many videos online like the linked one above. ThomasBoudier: How does 3D Density of 3D ImageJ Suite works What exactly is it calculating What do the different. My goal is to visually display the density map and quantify the location in the tissue with high concentration of the specific cells, eg based on distances from other objects. But I cannot contain myself to add my 2 cents to this topic, because some of those videos make me like… Hi, I’m looking for a way to compute cell density map, based on already segmented cells (in 2D). No offense to no one making those videos or taking them as orientation in case of the lack of other available resources. This was the one I looked at Analysing blots and gels with ImageJ/Fiji - YouTube
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