There is no one best way to select a threshold. Nor, for any given image, is there always a best threshold. For your images, you can see that you have a continuum of gray levels and the possibility of picking any gray level for the threshold. It's basically a judgement call. You can use an interactive threshold selection tool, like mine:
to pick one. So, if you pick one, who's to say it's wrong? Nobody. There is nothing in the physics of the situation that says what the best threshold is. Maybe there could be if you had an image of a perfectly black background (like black velvet which is a cheap super dark background to use), and all your droplets are known to be brighter than the background. Then you could just set your threshold to be the maximum gray level of your background. However if your background is not uniform, meaning it might be brighter in some areas than your droplets are in other areas, then that might not work.
You need to make sure your background is as dark and uniform as possible. Not sure if you know about lens shading, but all lenses shade the image so the the edges and corners of the image are usually darker than the middle (optic axis). You need to correct for that. The usual way is to divide your test images by the background image, though for certain situations (x-ray, fluoresence) you need to subtract instead of divide. For example, if the corner of your image is 90% as bright as the middle, you'd need to divide the image by 0.90 there to "undo" the effect of lens shading. This will also handle lighting non-uniformity. A demo is attached.
However you are correct in saying that the best way is to use a fixed threshold. Whatever it is chosen to be, use that threshold for all your images otherwise you can't accurately compare the images that had a lot of spray with those that had not much spray since the threshold would be different with every image.
If you want to use an automatic threshold, often times I find the triangle threshold works very well. I'm attaching it.
[pixelCounts, binEdges] = histcounts(grayImage, 255);
lowThresholdIndex = triangle_threshold(pixelCounts, 'R', false)
lowThreshold = binEdges(lowThresholdIndex)
highThreshold = binEdges(end)
[lowThreshold, highThreshold] = threshold(lowThreshold, highThreshold, grayImage)
mask = grayImage >= lowThreshold & grayImage <= highThreshold;
So once you've decided upon the threshold(s), via whatever method you choose, you need to use that value(s) for all your images.
