When reading a paper, how do you evaluate other peoples data? Do you look at an image and say, yes what you’re saying is obvious? Or do you look at the image and say, what you say appears obvious, but the quality of your image is not very good, so I don’t really trust it?
I am reading a paper that is quite important in my particular subfield. As I read the paper, I examined the microscopy images in detail as they were the first to demonstrate “colocalization” of my proteins of interest. IMHO, the images are not very good. As the authors used confocal microscopy, they either had too much ab, the detector pinhole set to > 1 airy disc or the pmt gain and offset settings were too high. They also stated that the 2 proteins colocalized without providing information on the voxel (a 3 dimensional pixel) size used to collect the images. Yes the 2 signals overlap, but is that because you used a 100 nm X 100 nm X 250 nm or because you used something greater than that? because if you used something bigger, I don’t care that signal from 2 proteins share the same space it doesn’t mean they interact. Heck signal from 2 proteins can occupy the same 100nm x 100 nm x 250 nm and that doesn’t mean they interact. In another set of images, as expected the images collected using GFP-tagged protein is much cleaner then images collected wheere an antibody was used to detect the protein. For those of you who don’t know, there will be additional noise with an antibody because of non-specific binding. It is for this reason, that is why you always take images of your cells, tissue, etc with no antibody (account for auto fluorescence), secondary only (non-specific ab binding) and / or an isotype control. I say and/or as I’ve heard legitimate arguments for an and against it – I just haven’t decided which side of the fence I sit on. These control images are used to remove “noise” or background (which should be stated in the methods section!!!)
As I explained my critique to a fellow lab member, they became somewhat defensive** and stated that when critiquing someone else’s data I should ask if it passes the bloody obvious question before slamming the quality of it. I don’t agree with this, because I know if MSc advisor had seen those images, he never would have let me publish them. Plus the images coming out of our lab do not look like that. Which brings me to my question for you dear readers? How do you evaluate data?
**although this individual was strongly annoyed with me (probably because they think I was being overly critical and they know how hard the first author worked), they were perfectly normal with me 10 minutes later. I love that we can be strongly opinionated on a subject and then be totally normal on the next topic.
5 hours ago