Before you do anything first decide whether the orders of magnitude difference in CS is going to be relevant. Procrustes analysis will uniformly scale the coordinates using the centroid size from each specimen, so they all will have unit size. So, from analytical point of view there is no harm if you are only going to use the procrustes aligned coordinates or PC derived from them in your analysis. The moment you will run into issues is when you include the actual centroid size as a variable in your analysis, whether for allometric regression or for simply using it species comparison. Imagine a case where you have two specimen of the same species, one with centroid size in thousand (because probably the coordinates are in raw pixel distances) versus one it is in tens (because probably same scaling has been already applied to the image either directly or indirectly). That obviously wouldn't make much biological sense.
If you had ruler visible in all your images, proper way to correctly scale the data is to measure a known distance (e.g., 50mm) in ImageJ and look at the length reported in pixels (e.g., 500 pix). Then the correct scale of data is 10pixels/mm (or in other words 100 micron resolution), which you can apply using the menu you have shown in your screenshot. This of course assumes the measured distance is more or less parallel to one of the image axes. The more off-axis it is, the more error you will make. If the field of view and the image resolution didn't change between acquisition, then you probably only have to do this on a few samples, and if they are all coming out around the same value, you can use that as your the scaling coefficient for the remaining samples without having to measure them. However, if the field of view has changed (zoom in/out), or you changed the resolution of the images after the acquisition you can't rely on your one size fits all type of scaling, and have to go one by one. There are three orders of magnitude difference in your centroid size plot, so it is hard to tell whether the clusters around thousands range are due to different image acquisition parameters, or natural size variation (which might be the case if you kept all the imaging parameters the same and your specimens vary quite a bit in size). All I can say, you definitely have minimum of two groups for sure (thousands range, and the ones close to zero). The rest you have to decide by trial and error. Again, if you are not planning to use centroid size anywhere in your analysis, then all this is moot. You can stick to procrustes coordinates based shape analysis, and all will be good. On Friday, October 18, 2024 at 3:40:27 PM UTC-7 Chris J Conroy wrote: > Hello morphologists, > > I’ve created a landmark dataset for ~250 mammal skulls. My process > was to take images in the same view, with a ruler in the image. I opened > the files in ImageJ and plotted the points. I’ve made an input file in > MorphoJ and done the usual analyses, which largely make phylogeographic > sense. However, centroid size appears to be in semi-discreet bands of > values ranging from near 0 to near 4000. I’m sure the variation comes from > at least two sources. One is that I used two cameras, one at 72 DPI and the > other at 96 DPI. Opening the images in Mac Preview allows me to check this. > Next, in ImageJ, it looks like for some of them I set a scale based on the > image density and others I did not. The landmark XY values themselves > differ by orders of magnitude. > > I took a sample of specimens from top to bottom of the centroid size > variation and fixed the scale in ImageJ and recreated the landmarks. The XY > values are now in the same range. > > Before I sit down and do this for all 250 images, I wanted to see if this > is going to be a waste of time, or will images from different digital > cameras going to be fundamentally different in centroid size and not worth > the work. I suppose I could do the work and then code all the images by > camera and see if there is an obvious effect. Surely I’m not the first > person to go through this. > > If it comes to it, I can re-take the lower res images on the higher > resolution camera. > > Lastly, on the higher resolution camera, some images are slightly zoomed > to fill the image with the object. Will this also mess up centroid size? > > Thanks, > > Chris > > > > -- You received this message because you are subscribed to the Google Groups "Morphmet" group. To unsubscribe from this group and stop receiving emails from it, send an email to [email protected]. To view this discussion on the web visit https://groups.google.com/d/msgid/morphmet2/9f109eaa-e205-4976-93ba-b295a509ffden%40googlegroups.com.
