Legitimate and illegitimate use of Photoshop® to prepare your data for publication

Recently, journals such as JCB have adopted an explicit policy with respect to the preparation of images for publication of experimental results (Rossner, M., and K.M. Yamada. 2004. What's in a picture? The temptation of image manipulation. J Cell Biol. 166:11). The purpose of this document is to describe two specific features in the policy that we must make special efforts to abide by.

While everyone agrees that sample misidentification, focal image manipulation (i.e. erasing and pasting bands, changing the background in one part of the image, etc.), are all illegitimate, the new rules regarding the legitimacy of applying uniform adjustments to the gain/brightness of an image might not be as intuitively obvious. Nonetheless, they are rigidly enforced. The new policy specifies that: “While it is acceptable practice to adjust the overall brightness and contrast of a whole image, such adjustments should not obscure or eliminate any information present in the original".

In his paper on the subject the executive editor at JCB states that: “It may be argued that this guideline is stricter than in the days before Photoshop, when multiple exposures were used to perfect the presentation of the data. Perhaps it is, but this is just one of the advantages of the digital age to the reviewer and editor, who can now spot these manipulations when in the past an author would have taken the time to do another exposure.” He urges us to “think about this when you are doing the experiment and perform multiple exposures to get the bands at the density you want” (Rossner & Yamada, 2004). For example, when performing an immunoblot it is legitimate and indeed desirable to choose the amount of protein loaded on the gel, the concentration of antisera, the incubation time and the time of exposure to ECL reagent; all to maximize the signal to noise ratio and to avoid background signal (biological or artifactual). But the printed version of the experiment that will be sent for review must contain all the information that was on the best film, to do otherwise is scientific misconduct!

This might seem a somewhat arbitrary injunction: ECL is a non-linear signal acquisition technique; why then is it legitimate to arrange all the “wet” parameters of the experiment to access a narrow (and often difficult to reproduce) detection threshold that maximizes signal to noise and illegitimate to set, post-hoc, the threshold at which the result is processed for publication so as to focus attention on the same signal. After all, scientific thinking selects signal from noise and focuses on the former. However, the implicit position of the Journal is that images are NOT quantitative data (on which one can set the gain) but rather a unique item of evidence acquired at one specific moment and admitted to the court of peer review. As experimentalists we are trained to ignore irrelevant data (this is crucial to developing theories); we look at the parameter that changes in a reproducible and interpretable manner and ignore the distracting and uninterpretable background. The new rules say that we can design every aspect of our experiments to eliminate the latter, but we cannot use Photoshop to that effect. While this might seem a grey zone in the mind of some (I suspect that casting agencies have similar problems with actor’s head-shots), it is viewed as a black and white issue by the Journal editors and policymakers. Please look at figure 3 in the online article to get a vivid impression of what the rules mean to us.

A second point pertains to the grouping of images from different parts of the same gel. This must be made explicit by the arrangement of the figure (e.g., using dividing lines), and must also be made explicit in the text of the figure legend (e.g. “lanes 1-13 and 14-18 were grouped together from separate immunoblots, developed together”).

More than honor is at stake. JCB now only accepts for publication images that admit sufficient background to safeguard against the practice of excessive adjustment. They routinely survey the images for such and they threaten violators of their policy with severe sanctions. Furthermore, these criteria are bieng adopted by other journals and funding agencies. Therefore, before submitting a paper to any journal, print out the images you want to submit and compare them side-by-side with the films on which they are based. Please do this first yourself and then review the same together with me, or, if I am away with another senior colleague (preferably one who is not a co-author on the paper).

Research requires a tremendous effort and is often conducted at great personal expense; it is tragic to have hard won truths invalidated by failure to adhere to these rules.

David Ron

December 4, 2005