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Information on this page assumes Windows OS, Adobe Photoshop version 23.0 (Windows).

Read and understand "Information for Authors" for your intended Journal

Every journal has its own figure format, so pay attention to what they are asking for. Not following the instructions can result in published figures with a sub-optimal resolution, shifted colors, shifted brightness/contrast, etc., etc. You've worked so hard to get to a point where you are ready to publish your interesting data, right? So let's make sure to show your data to the world in the best way possible!

In general, most journals will have specific requirements for the following: file format, figure dimensions, color mode and resolution, use of colors, figure annotations.

So let's start by looking at an example and figure out what they are talking about. Below is a set of instructions for figures to be published in one of our favorite journals, Journal of Neuroscience (Information as of Dec. 2021; bold by MK).

Figures

Figures must be numbered independently of tables, multimedia, and 3D models and cited in the text. Do not duplicate data by presenting it both in the text and in a figure.

A title should be part of the legend and not lettered onto the figure. A legend must be included in the manuscript document after the reference list, and should include enough detail to be intelligible without reference to the text.

Figures must be submitted as separate files in TIFF or EPS format and be submitted at the size they are to appear: 1 column (maximum width 8.5 cm), 1.5 columns (maximum width 11.6 cm) or 2 columns (maximum width 17.6 cm). They should be the smallest size that will convey the essential scientific information.

Illustrations should be prepared so that they are accessible to our many color-blind readers, so color should only be used if it is necessary to accurately convey the information being presented by the image. Grayscale generally provides a more faithful representation when a single quantity is displayed. Use textures or different line types rather than colors in bar plots or graphs. Figures with red and green are particularly problematic, and should generally be converted to magenta and green. If no suitable combination can be found, consider presenting separate monochrome images for the different color channels. For line drawings that require color, consider redundant coding by adding different textures or line types to the colors.

Color figures should be in RGB format and supplied at a minimum of 300 dpi. Monochrome (bitmap) images must be supplied at 1200 dpi. (← MK note: they are talking about graphs and line drawings in raster file format here.) Grayscale must be supplied at a minimum of 300 dpi. For figures in vector-based format, all fonts should be converted to outlines and saved as EPS files to ensure that they are reproduced correctly.

Remove top and right borderlines that to not contain measuring metrics from all graph/histogram figure panels (i.e., do not box the panels in). Do not include any two-bar graphs/histograms; instead state those values in the text.

All illustrations documenting results must include a bar to indicate the scale. All labels used in a figure should be explained in the legend. The migration of protein molecular weight size markers or nucleic acid size markers must be indicated and labeled appropriately (e.g., “kD”, “nt”, “bp”) on all figure panels showing gel electrophoresis."

So what does JNeurosci ask for?

File formatTIFF or EPS
Figure dimensions1, 1.5, or 2 columns
Color mode and resolutionRGB color or grayscale at 300 dpi; monochrome at 1200 dpi
Use of colorsMake them color-blind friendly!
Figure annotationsscale bars, MW size markers, etc.

File format: Raster vs. Vector

There are two categories of digital artwork: Raster and Vector images. Raster images are pixel-based files like EM images, and they include formats like TIFF, PNG, JPEG, BMP, and PSD. To edit a raster file, you would be using tools like Fiji/ImageJ, Adobe Photoshop, and GIMP. Vector images are object-based files like graphs created in Microsoft Excel and GraphPad Prism, and these images are supported by file formats including EPS, PDF, SVZ, PPTX, and AI. To work with a vector file, you are probably using Adobe Illustrator, Adobe InDesign, Microsoft PowerPoint, or Inkscape (there are others as well). In general, most journals prefer vector files because they can ensure the highest quality and produce the best results in publication.

Figure dimensions

Figure dimensions are defined by typesetting format for the final PDF (or physical) print, and journal-specific. If your figure is not conformed to the specified dimensions, the journal's typesetter will have to resize it. This often results in loss of resolution in publication.

Color mode and resolution

Most journals will ask you to present your color images in RGB (or more precisely, sRGB) because it is the standard color space for displaying images online. Some print journals might talk about converting your files from RGB to CMYK, which is an ink-based color space and used for printing. Because RGB color space is wider ( i.e., capable of producing many more colors) than CMYK, there will be a color shift when images are converted from RGB to CMYK for print. That is, if a RGB color falls outside of CMYK space, then it will be re-mapped to fit inside the CMYK color space. There are ways to ensure the best possible conversion for CMYK print, and that is a topic for another discussion.

Resolution is set to the number (or sometimes range of numbers) that ensure the best results for publication. For most practical purposes, "dpi" (dots per inch) and "ppi" (pixels per inch) are interchangeable. If you are submitting your figures in a raster format like TIFF, following the resolution specification is critical because it has a direct consequence on how sharp your figures will look in publication. If you are making your figures in a vector format, it is important to follow the specification for halftone (RGB color or grayscale) images.

Your figure may contain a set of grayscale serial EM images, a couple of color 3D reconstruction images in RGB color, and several graphs. This figure would be considered as "combination halftones" because you have both raster and vector images in a single file. Unfortunately, JNeurosci tells you what resolution to use only for halftones (grayscale and color) and monochrome (line drawings), but not the mix of both. So what to do? If you are making your figure as a TIFF (i.e., raster) file, a good rule of thumb is to go for 900 ppi. Some journals will give you a specific number for this type of figure, so RTFM for your intended journal.

Another thing to remember when you submit your figure as a raster file is that your journal may have a limit for file size. This may become an issue when you have a large (e.g., a full page size) figure at a high resolution for line drawings or combination halftones. Some journals allows for TIFF files to be compressed in LZW and this could help. In any case, this is another reason you probably want to use software that support vector images.

For some journals (e.g., eLife), you will be asked to embed the figures within the text, at least for initial submissions. If this is the case, assemble your figures using software that support vector images, then export them as raster files (PNG works well for this) at resolution needed for a given type of the figure.

Use of colors

If you are using colors to represent specific experimental conditions, make sure they are consistently applied throughout your paper (e.g., LTP = red; control = blue).

Many journals do ask you to make your figures accessible to readers with colorblindness. See this and this for more details.

Figure annotations

Scale bars are required when you use microscope (or other) images as part of your results.

The font size should be big enough for all annotations to be legible. The smallest font should be 7 points or so (some journals will give you specific sizes). Use the same font across all figures.


Story-boarding

Decide which piece of data to present in what order: What is your paper about? What is the most logical and coherent order to present your data? Which images/graphs are critical for following and understanding key points of the paper?

Understand constraints of your intended journal: How many figures are you allowed to include in the main paper? Are you allowed to include any supplemental figures? If so, how many and in what format?

Plan each figure: How would you layout each figure? How big will each image/graph be?


Editing raster images non-destructively using Adobe Photoshop

Notes on image manipulation

Powerful image editing software tools like Adobe Photoshop make it so easy to adjust or modify image files. But you as a scientist need to know what constitutes acceptable vs. inappropriate changes to your original data. Remember that manipulated images may not necessarily affect your study's findings, but they are still considered as a form of misconduct. Some journals inspect all digital images in accepted manuscripts by specialists for any manipulations that may fit their guidelines for scientific misconduct (an example here).

It's just not worth it – if you feel the desire to manipulate images to fit your story, then maybe your story just doesn't hold water all that well. I know you've worked on this story for so long and you feel so attached and it's like your child. You might even feel your career depends on it. (But really, you want to base your career on something like that?) I want you to direct your time and energy to get to a place where you can just accept holes and loose ends, and make them open so that your audience could get a more complete picture of your interesting study. I would say this is better than publishing results that no one can reproduce in a high profile journals. I would also say that this gives you an opportunity to get to know your data better and get you to think about the next set of experiments. So go back to your drawing board. Talk to your colleagues. Go over your data again with your advisor. Just do the right thing, OK?

Now here's your reading assignment. Read this paper before you start working on your images. It is a bit dated, but still provides good guidelines and is a great starting point in thinking about this issue.

Pixel size

Before you start, you need to know (where to find) pixel size of your image. If you are using an image from a 3DEM series in Reconstruct, this information can be found in domains. If pixel size was calibrated/corrected during analysis, the pixel size listed in domains may not be correct. If this is the case, Read this page.

Color space

Adobe Photoshop is capable of handling different flavors of RGB color space, including sRGB, Adobe RGB, and ProPhotoRGB. Adobe RGB and ProPhotoRGB is wider than sRGB, and therefore, they can make more colors than sRGB. I know that sounds nice, but currently, sRGB is the default standard for displaying images online. And the vast majority of computer monitors (including yours in the lab) can display sRGB only. So it would make sense to start with your tool set to handle sRGB. To do this:

  1. Go to "Edit" > "Color Settings" (Shift+Ctrl+K)
  2. In the Color Settings window under "Working Spaces", choose "sRGB IEC61966-2.1" for "RGB".
  3. Click "OK".
Color Settings window

Notes on 3DEM images

When you prepare a 3DEM image for a figure, use the original, pre-alignment image whenever possible. This is because the aligned images can exhibit local stretching etc. that degrades image quality.

Open an image file and making its copy

  1. Go to "File" > "Open" (Ctrl+O)
  2. Navigate to the folder that contains your image.
  3. Click on the image to be edited. (You can hold Ctrl while clicking to choose multiple images.)
  4. Click "Open".
  5. Go to "File" > "Save As..." (Shift+Ctrl+S)
  6. Enter a new name for this image file under "File name".
  7. Choose "Photoshop (*.psd)" for "Save as type:".
  8. Click "Save".

Adjusting Image size

By now, you should know what resolution your halftone images should have – so let's make sure your images set to the correct resolution while preserving all pixels in them.

  1. Go to "Image" > "Image Size" (Alt+Ctrl+I)
  2. In the "Image Size" window,
    1. Uncheck "Resample"
    2. Under"Resolution, Enter "300". Make sure "Pixels/Inch" is selected.
    3. Click "OK".
"Image Size" window

Cropping

This example image is 13184 × 9808 pixels, so at 300 ppi, it is 43.947 × 32.693 inches. I know my final image size should be ~4 × 3 inches, but that can change depending on how images are arranged in the figure. So it would be great if we could crop the image to an approximate size, but also have some flexibility for finer adjustment later. Here's a way to crop an image without throwing away any pixels:

Click Crop tool (keyboard shortcut = c). Now you should see your image surrounded by the crop handles.

If you know the aspect ratio of the final image, enter the numbers in the crop menu (next to "Ratio"). I'm keeping them blank for this example.

Uncheck "Delete Cropped Pixels" in the crop menu.

Click and drag any of the crop handles to adjust the crop area size.

Click and drag the underlying image to adjust its position in the crop area.

If you need to zoom in closer, hit Ctrl+"+". To zoom out, Ctrl+"-".

If you want to start over, hit Esc. The crop handles will go back to edges of the image.

When ready to crop, right click in the crop area and choose "Crop".

Save the image (Ctrl+S).

If you (or your PI) changed your mind and want to make a different crop, simply go back to the crop tool, and clock on the image. The crop handles and the entire image will re-appear.

Crop handleCrop menu


Ready to crop...Cropped!

Adjusting brightness and contrast

Understanding histogram


Export the final image


Formatting graphs

Know the final image size

Output from Excel: copy and paste into Adobe Illustrator or PowerPoint

Output from Prism: export as EPS, then open in Illustrator

Putting together a figure using Adobe Illustrator

Assuming Adobe Illustrator version 26.0 (Windows)

Setting up a file

Go to "File" > "New"

In the "new Document" window, enter the following: title (default is "Untitled-1"), width and height (If you don't know the final size, leave it as the Letter size - 8.5 x 11 in), color mode (choose "RGB color", ), raster effects (choose "High (300 ppi)"

A couple of things I find useful:

  • Rulers: "View" > "Rulers" > "Show Rulers". You can change the unit of the rulers by right clicking on a ruler.
  • Grids: "View" > "Show Grid". If your Rulers are in inches but the grid is not, you can change the grid here: "Edit" > "Preferences" > "Guides & Grid". Set "Gridline every:" to 1 in and "Subdivisions:" to 8.

Organize your images into layers

Your figures will probably have multiple images and graphs.

Placing images

Editing raster images

Editing graphs (vector images)

Basic drawing

Annotations: text, scale bars,

Clipping mask

Checking colors for colorblindness: "View" > "Proof Setup" > "Color Blindness" – There are two options. Check both to see how your figure appears. Can you differentiate different colors in your figure?

Convert text to outlines: this should be done for the final version

But I want to use Microsoft PowerPoint to assemble my figures!

Yes, I hear you. You've used PowerPoint all your life and you know it like back of your hand. And yes, you can insert raster images and graphs from Excel as vector images. In fact, an Excel graph pasted into a PowerPoint file will automatically update when changes are made to the source spreadsheet (as long as the link is maintained), so this can be very convenient.

Just like when using Adobe Illustrator, make sure that your raster images satisfy resolution requirements for your journal before inserting into PowerPoint.

There are things PowerPoint is not able to do, so you need to be aware of them:

  1. PowerPoint will not convert text into outlines. So If you have any special characters (like Greek letters and other non-English alphabets) in your figure, they may not be recognized by some computers and/or printers. This can be prevented if you copy and paste those special characters from Character Map in Windows.
  2. PowerPoint will not save as or export to an EPS file, so this would be an issue if your journal requires EPS format.
  3. PowerPoint will not proof your figures for colorblindness. Boo. (Just make sure to define your color palette beforehand.)
Character Map