Creating clear and informative image-based figures for scientific publications

Abstract

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

Fig 1. Image types and reporting of scale information and insets.

(A) Microscope images and photographs were common, whereas other types of images were used less frequently. (B) Complete scale information was missing in more than half of the papers examined. Partial scale information indicates that scale information was presented in some figures, but not others, or that the authors reported magnification rather than including scale bars on the image. (C) Problems with labeling and describing insets are common. Totals may not be exactly 100% due to rounding.

Fig 2. Use of color and annotations in image-based figures.

(A) While many authors are using colors and labels that are visible to colorblind readers, the data show that improvement is needed. (B) Most papers explain colors in image-based figures; however, explanations are less common for the species and tissue or object shown, and labels and annotations. Totals may not be exactly 100% due to rounding.

Fig 3. Selecting magnification and using insets.

(A) Magnification and display detail of images should permit readers to see features related to the main message that the image is intended to convey. This may be the organism, tissue, cell, or a subcellular level. Microscope images [18] show D. melanogaster ovary (A1), ovarian egg chamber cells (A2), and a detail in egg chamber cell nuclei (A3). (B) Insets or zoomed-in areas are useful when 2 different scales are needed to allow readers to see essential features. It is critical to indicate the origin of the inset in the full-scale image. Poor and clear examples are shown. Example images were created based on problems observed by reviewers. Images show B1, B2, B3, B5: Protostelium aurantium amoeba fed on germlings of Aspergillus fumigatus D141-GFP (green) fungal hyphae, dead fungal material stained with propidium iodide (red), and acidic compartments of amoeba marked with LysoTracker Blue DND-22 dye (blue); B4: Lendrum-stained human lung tissue (Haraszti, Public Health Image Library); B6: fossilized Orobates pabsti [19].

Fig 4. Using scale bars to annotate image size.

Scale bars provide essential information about the size of objects, which orients readers and helps them to bridge the gap between the image and reality. Scales may be indicated by a known size indicator such as a human next to a tree, a coin next to a rock, or a tape measure next to a smaller structure. In microscope images, a bar of known length is included. Example images were created based on problems observed by reviewers. Poor scale bar examples (1 to 6), clear scale bar examples (7 to 12). Images 1, 4, 7: Microscope images of D. melanogaster nurse cell nuclei [18]; 2: Microscope image of Dictyostelium discoideum expressing Vps32-GFP (Vps32-green fluorescent protein shows broad signal in cells) and stained with dextran (spotted signal) after infection with conidia of Aspergillus fumigatus; 3, 5, 8, 10: Electron microscope image of mouse pancreatic beta-islet cells (Andreas Müller); 6, 11: Microscope image of Lendrum-stained human lung tissue (Haraszti, Public Health Image Library); 9: Photo of Arabidopsis thaliana; 12: Photograph of fossilized Orobates pabsti [19].

Fig 5. Image types and their accessibility in colorblind render and grayscale mode.

Shown are examples of the types of images that one might find in manuscripts in the biological or biomedical sciences: photograph, fluorescent microscope images with 1 to 3 color hues/LUT, electron microscope images. The relative visibility is assessed in a colorblind rendering for deuteranopia, and in grayscale. Grayscale images offer the most contrast (1-color microscope image) but cannot show several structures in parallel (multicolor images, color photographs). Color combinations that are not colorblind accessible were used in rows 3 and 4 to illustrate the importance of colorblind simulation tests. Scale bars are not included in this figure, as they could not be added in a nondistracting way that would not detract from the overall message of the figure. Images show: Row 1: Darth Vader being attacked, Row 2: D. melanogaster salivary glands [18], Row 3: D. melanogaster egg chambers [18], Row 4: D. melanogaster nurse cell nuclei [18], and Row 5: mouse pancreatic beta-islet cells. LUT, lookup table.

Fig 6. Visibility of colors/hues differs and depends on the background color.

The best contrast is achieved with grayscale images or dark hues on a light background (first row). Dark color hues, such as red and blue, on a dark background (last row), are least visible. Visibility can be tested with mock grayscale. Images show actin filaments in Dictyostelium discoideum (LifeAct-GFP). All images have the same scale. GFP, green fluorescent protein.

Fig 7. Color combinations as seen with normal vision and 2 types of colorblindness.

The figure illustrates how 4 possible color combinations for multichannel microscope images would appear to someone with normal color vision, the most common form of colorblindness (deuteranopia), and a rare form of color blindness (tritanopia). Some combinations that are accessible to someone with deuteranopia are not accessible to readers with tritanopia, for example, green/blue combinations. Microscope images show Dictyostelium discoideum expressing Vps32-GFP (Vps32-green fluorescent protein shows broad signal in cells) and stained with dextran (spotted signal) after infection with conidia of Aspergillus fumigatus. All images have the same scale. GFP, green fluorescent protein.

Fig 8. Strategies for making 2- or 3-channel microscope images colorblind safe.

Images in the first row are not colorblind safe. Readers with the most common form of colorblindness would not be able to identify key features. Possible accessible solutions are shown: changing colors/LUTs to colorblind-friendly combinations, showing each channel in a separate image, showing colors in grayscale and inverting grayscale images to maximize contrast. Solutions 3 and 4 (show each channel in grayscale, or in inverted grayscale) are more informative than solutions 1 and 2. Regions of overlap are sometimes difficult to see in merged images without split channels. When splitting channels, scientists often use colors that have low contrast, as explained in Fig 6 (e.g., red or blue on black). Microscope images show D. melanogaster egg chambers (2 colors) and nurse cell nuclei (3 colors) [18]. All images of egg chambers and nurse cells respectively have the same scale. LUT, lookup table.

Fig 9. Planning multipanel figures.

Planning tables and layout sketches are useful tools to efficiently design figures that address the research question. (A) Planning tables allow scientists to select and organize elements needed to answer the research question addressed by the figure. (B) Layout sketches allow scientists to design a logical layout for all panels listed in the planning table and ensure that there is adequate space for all images and graphs.

Fig 10. Using arrows, regions of interest, lines, and letter codes to annotate structures in images.

Text descriptions alone are often insufficient to clearly point to a structure or region in an image. Arrows and arrowheads, lines, letters, and dashed enclosures can help if overlaid on the respective part of the image. Microscope images show D. melanogaster egg chambers [18], with the different labeling techniques in use. The table provides an overview of their applicability and common pitfalls. All images have the same scale.

Fig 11. Different levels of detail for image annotations.

Annotations help to orient the audience but may also obstruct parts of the image. Authors must find the right balance between too few and too many annotations. (1) Example with no annotations. Readers cannot determine what is shown. (2) Example with a few annotations to orient readers to key structures. (3) Example with many annotations, which obstruct parts of the image. The long legend below the figure is confusing. (4) Example shows a solution for situations where many annotations are needed to explain the image. An annotated version is placed next to an unannotated version of the image for comparison. The legend below the image helps readers to interpret the image, without having to refer to the figure legend. Note the different requirements for space. Electron microscope images show mouse pancreatic beta-islet cells.

Fig 12. Explain color in images.

Cells and their structures are almost all transparent. Every dye, stain, and fluorescent label therefore should be clearly explained to the audience. Labels should be colorblind safe. Large labels that stand out against the background are easy to read. Authors can make figures easier to interpret by placing the color label close to the structure; color labels should only be placed in the figure legend when this is not possible. Example images were created based on problems observed by reviewers. Microscope images show D. melanogaster egg chambers stained with the DNA dye DAPI (4′,6-diamidino-2-phenylindole) and probe for a specific mRNA species [18]. All images have the same scale.

Fig 13. Annotations should be colorblind safe.

(1) The annotations displayed in the first image are inaccessible to colorblind individuals, as shown with the visibility test below. This example was created based on problems observed by reviewers. (2, 3) Two colorblind safe alternative annotations, in color (2) and in grayscale (3). The bottom row shows a test rendering for deuteranopia colorblindness. Note that double-encoding of different hues and different shapes (e.g., different letters, arrow shapes, or dashed/nondashed lines) allows all audiences to interpret the annotations. Electron microscope images show mouse pancreatic beta-cell islet cells. All images have the same scale.