Guide for digital images

Images submitted with a manuscript for review should be minimally processed (for instance, to add arrows to a micrograph). Authors should retain their unprocessed data and metadata files, as editors may request them to aid in manuscript evaluation. If unprocessed data are unavailable, manuscript evaluation may be stalled until the issue is resolved. All digitized images submitted with the final revision of the manuscript must be of high quality and have resolutions of at least 300 dpi.

A certain degree of image processing is acceptable for publication (and for some experiments, fields and techniques is unavoidable), but the final image must correctly represent the original data and conform to community standards. The guidelines below will aid in accurate data presentation at the image processing level; authors must also take care to exercise prudence during data acquisition, where misrepresentation must equally be avoided. Manuscripts should include a single Supplementary Methods file (or a subsection of a larger Supplementary Methods file) labeled �equipment and settings� that describes for each figure the pertinent instrument settings, acquisition conditions and processing changes, as described in this guide.

  1. Authors should list all image acquisition tools and image processing software packages used.
  2. Authors should document key image-gathering settings and processing manipulations in the Supplementary Methods.
  3. Images gathered at different times or from different locations should not be combined into a single image, unless it is stated that the resultant image is a product of time-averaged data or a time-lapse sequence. If juxtaposing images is essential, the borders should be clearly demarcated in the figure and described in the legend.
  4. The use of touch-up tools, such as cloning and healing tools in Photoshop, or any feature that deliberately obscures manipulations, is to be avoided.
  5. Processing (such as changing brightness and contrast) is appropriate only when it is applied equally across the entire image and is applied equally to controls. Contrast should not be adjusted so that data disappear. Excessive manipulations, such as processing to emphasize one region in the image at the expense of others (for example, through the use of a biased choice of threshold settings), is inappropriate, as is emphasizing experimental data relative to the control.
  6. When submitting revised final figures upon conditional acceptance, authors may be asked to submit original, unprocessed images.

Electrophoretic gels and blots

Positive and negative controls, as well as molecular size markers, should be included on each gel and blot � either in the main figure or an expanded data supplementary figure. For previously characterized antibodies, a citation must be provided. For antibodies less well characterized in the system under study, a detailed characterization that demonstrates not only the specificity of the antibody, but also the range of reactivity of the reagent in the assay, should be published as supplementary information.

The display of cropped gels and blots in the main paper is encouraged if it improves the clarity and conciseness of the presentation. In such cases, the cropping must be mentioned in the figure legend and the supplementary information should include full-length gels and blots wherever possible. These uncropped images should be labeled as in the main text and placed in a single supplementary figure. The manuscript�s figure legends should state that �full-length blots/gels are presented in Supplemental Figure X."

  • Vertically sliced gels that juxtapose lanes that were not contiguous in the experiment must have a clear separation or a black line delineating the boundary between the gels.
  • Cropped gels in the paper must retain important bands.
  • Cropped blots in the body of the paper should retain at least six band widths above and below the band.
  • High-contrast gels and blots are discouraged, as overexposure may mask additional bands. Authors should strive for exposures with gray backgrounds. Multiple exposures should be presented in supplementary information if high contrast is unavoidable. Immunoblots should be surrounded by a black line to indicate the borders of the blot, if the background is faint.
  • For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used.

Microscopy

Authors should be prepared to supply the editors with original data upon request, at the resolution collected, from which their images were generated. Cells from multiple fields should not be juxtaposed in a single field; instead multiple supporting fields of cells should be shown as supplementary information.

Specific guidelines: Adjustments should be applied to the entire image. Threshold manipulation, expansion or contraction of signal ranges and the altering of high signals should be avoided. If �Pseudo-coloring� and nonlinear adjustment (for example �gamma changes�) are used, this must be disclosed. Adjustments of individual color channels are sometimes necessary on �merged� images, but this should be noted in the figure legend.

We encourage inclusion of the following with the final revised version of the manuscript for publication:

  • In the Methods, specify the type of equipment (microscopes/objective lenses, cameras, detectors, filter model and batch number) and acquisition software used. Although we appreciate that there is some variation between instruments, equipment settings for critical measurements should also be listed.
  • A single Supplementary Methods file (or subsection of a Supplementary Methods file) titled �equipment and settings� should list for each image: acquisition information, including time and space resolution data (xyzt and pixel dimensions); image bit depth; experimental conditions such as temperature and imaging medium; and fluorochromes (excitation and emission wavelengths or ranges, filters, dichroic beamsplitters, if any).
  • The display lookup table (LUT) and the quantitative map between the LUT and the bitmap should be provided, especially when rainbow pseudocolor is used. If the LUT is linear and covers the full range of the data, that should be stated.
  • Processing software should be named and manipulations indicated (such as type of deconvolution, 3D reconstructions, surface and volume rendering, 'gamma changes', filtering, thresholding and projection).
  • Authors should state the measured resolution at which an image was acquired and any downstream processing or averaging that enhances the resolution of the image.