Tuesday, September 29, 2015

Stacking the deck with images?

I wish that I could solicit input on this question as an unbiased observer. Anyone who's read my previous posts knows that I am anything but. I considered presenting this data absent the source, but anyone with 1% of Sherlock Holmes' sleuthing ability would track down the paper I am referring to in a nanosecond.  Going back and forth with the EIC at ACS Applied Materials and Interfaces recently (maybe more on this later) forced me to look at aspects of this paper in greater detail than I would have otherwise. As I documented before, one of the reviewers fixated on the cell imaging aspects as a defense of the results, and a rationale to reject my Comment. In preparing an appeal, I looked more carefully at the cell images

Here are cells treated with Fe3+ and probe:

The difference between a) and b) is 1 hr of incubation time. Ref: ACS Appl. Mater. Interfaces20146, 18408–18412

Here is the control of cells treated only with probe:
The difference between d) and e) is 1 hr of incubation time. Ref: ACS Appl. Mater. Interfaces20146, 18408–18412

Notice any differences? I am not very experience with growing cells, but to me it looks like the confluency is much higher and the cell size is larger in the images of the iron-treated cells (Figure a-c), than the sensor-only cells (Figure d-f). This could create the illusion of a greater fluorescence response in the iron-treated cells. There is simply more sensor present per unit area, and there is clearly basal fluorescence. In fact, I might argue that the sensor-only cells (f) are not even healthy compared to the Fe3+-treated cells (c). It also appears that the fluorescence images of the sensor-only cells are out of focus. Even the brighter cells in the middle of the field look fuzzy. Again, this could make the response look more dramatic than the reality. There are other problems with this experiment related to metal transport, but I don't want to conflate imaging with biology. I have a biological interpretation backed by literature for what they observe in the Fe3+-treated cells.

I am interested if anyone concurs, or if I am the victim of confirmation bias. There have been several notorious examples of image manipulation including cut & paste to make nanochopsticks and reusing old data. This isn't that kind of malfeasance, but could it be stacking the deck to fit a preconceived model? How say you?

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