Appropriate Image Manipulation in Scientific Publishing

1. Moderate, linear changes applied to the entire image. Linear changes mean brightness, contrast, and color balance revisions. Any change should be applied to the whole image, not a local area. All changes should also be moderate so that they do not cause changes in the interpretation of the results. For example, if you increase the brightness too much, you might make too many pixels have the maximum brightness and therefore lose the subtle differences among these pixels (Fig. 1). Avoid using an image program’s auto contrast, auto levels, and auto color tools, as these may overprocess your image.
Adjusting the gamma value is considered a non-linear change and should be avoided. When gamma value adjustments and pseudo-coloring are necessary, they should be disclosed in the paper. Another point to keep in mind is that if you have a group of images to be shown together for comparison (e.g., a control vs. several different treatments), all the pictures should be taken using the same setting and manipulated in the same way. Yes, this means you should have a record of the setting(s) used for photographing and the way you manipulated the images. It is recommended this be prepared in advance.
2. Cropping. We often do not need to show the entire picture we receive from the camera or scanner, so it is generally allowed to crop an image to remove unnecessary information or empty space around the edges. However, cropping should not alter the results or introduce ambiguity. Let’s again use the western blot in Fig. 1 as an example. While it is customary to show only the protein band of interest, removing an unwanted band by cropping is not correct. Cropping too close to the band may make readers suspect that you are trying to cover up the fact that there is another band nearby (Fig. 2). Some journals require that a certain area of the blot above and below the band must be shown in the figure.
After cropping, if you need to stitch some image pieces together, the pieces should be outlined (or separated by some white space) to clearly show that they are from different sources (Fig. 3).
3. Re-sizing. An image published in a journal article is almost never the same size as the original picture. Re-sizing is a necessary step to ensure your figure meets the journal-desired size. It is generally acceptable to make an image smaller (i.e., decreasing the number of pixels). Computer software can combine multiple pixels into one pixel by averaging their values, and the human eye will not detect a difference. However, do not try to increase the number of pixels of an image, because the computer software will have to create extra pixels that are not already there. This causes misinterpretation of your data and does not make a low-resolution image look better.
Before re-sizing, a scale bar should be added to the image so that you have a correctly sized scale bar for the final figure. A group of images to be shown together for comparison should be re-sized the same way.

1. Plan ahead. If you have planned your experiments well, you are less likely to find it necessary to rearrange or manipulate your figure(s) for publication.
2. Take the best pictures. Get familiar with the instrument and software you use for image acquisition, so that the pictures you take are close to the way they should appear in your paper. Take pictures at the highest resolution possible and save in the correct format (TIFF, not JPEG). You should also save files in the native file format of the image acquisition software because such files may contain metadata of instrument settings such as the magnification information.