Tips for Illustrating Biological Pathways

Overview

Welcome to the BioRender’s Learning Hubs Design Tips for Industry Scientists. Today's focus will be on illustrating biological pathways, a very special type of figure that I think a lot of us sometimes dread making. And, kind of rightly so, they are quite intimidating to put together. But we've broken it down into general concepts and design tips, so hopefully, it'll be easy for you to tackle your next one.

For those that have never used BioRender before, it looks like we have a few new faces or people that have not tried or signed up for the app yet. It's a really easy-to-use, drag-and-drop illustration tool, so everything that you need, hopefully, is in the library, ready for you to drag and drop onto the canvas so you can create things like biological pathways, protocols, timelines, cell-to-cell interactions, all those kinds of things to help you communicate your science.

And, of course, some common use cases for Biorender include presentations foremost, I would say, for industry scientists, many of you can relate to that. Publications, of course, grant applications if you're doing that, proposals, posters, if I'm going to get back to that one day for conferences, experimental outlines, and even speaking to the lay audience. I know lay summary figures are becoming more and more popular as well. You want to be able to communicate your science to the general public, so figures, of course, are a really great way to do that.

Alright, so let's get started. We have a lot of tips that we've consolidated based on our over decade of making signaling pathways, intracellular mechanisms, and method of action. Hopefully, whatever we show you today, you'll be able to implement in your next figure.

The reason why biological pathways or pathway diagrams or signaling pathways are so complex and intimidating to approach is that we're used to seeing these kinds of images. Obviously, they serve different purposes depending on the level of complexity and the output, but generally speaking, they end up being a lot of these circles with words in them and then criss crossing arrows. They are intimidating to draw and also intimidating to consume the information as a viewer.

So that's probably what you're familiar with seeing. Here's another example of a sort of pathway diagram, and there's definitely a reason why it starts to get very complex. Obviously, nature itself is very, very complex, way more complex than this is even showing, and also this is not what you would see with the naked eye if you were to look at that magnitude. So there's a lot of abstraction happening, a lot of artistic freedom that we take when we represent structures and entities and pathways. It ends up being in our hands to be able to communicate it very clearly, succinctly, and also in a standardized way.

Of course, there's also these kinds of images that are almost like a poster. I've seen these a lot if I walk into a lab and on the wall, a lot of you may have these as almost decorative. It doesn't necessarily serve a purpose for publishing in a paper, for example, but maybe it'll be earmarked or bookmarked or something if you want to refer to it almost like an encyclopedia or again, a little bit more artistic in its rendering. I kind of equate this to something similar to a map or more of an artistic map. So this is the wine regions of France. I kind of pulled this off of the web, but you know, very beautiful, kind of a cool thing to print and put up on my wall. But I suspect if you are in the south of France, you'd probably want more of a directional image like this, you know, how to get from point A to point B, less so about what is all the information possible in the most beautiful way rendered in the most decorative form.

So I like to think of building out pathway diagrams, or really any scientific diagram, in this way. So really stripping away what you don't need to show with what you do need to show or what is important, what's nice to have, what versus what is needed to have. Sometimes we have to make those sacrifices by dialing back the story to show what is necessary and what is not okay.

With that, we've kind of distilled our learnings into four tips for today, just for the purposes of time and of course, how much information you can probably consume in one Zoom session. We've distilled it to these four concepts. One is optimizing the flow of information. It really goes for any kind of figure, so even if you're not making biological pathways, this will definitely apply to any kind of science figure you're making. Two is the idea of color. This is not really a tip but general concepts around that, which is proper use of saturation, contrast, which all relates to not only legibility but really servicing all types of users, including those that are colorblind. And number three is zooming in to show the correct scale. There's a couple of tricks here that you can use as far as orienting your viewer so that they're not constantly zooming in and zooming out from the macro-model organism level to the cell to the tissue to the proteins. There's a way to show that a little bit more succinctly. And then four is the proper use of lines and arrows, as you saw and have experienced.

Biological pathways involve arranging basic elements such as squares, shapes, arrows, and lines in a specific way. There are some general rules to follow that will help clean up your figure and make it less chaotic. Optimizing the flow of information is important regardless of the type of figure you're making. There are a few basic information flows or compositions to adhere to, such as unidirectional left to right, up to down, or cyclical. Avoid going in the opposite direction. The principle of proximity is also important for pathways. When putting together concepts, they should be easy enough to move around on your canvas as if they were stuck to a sticky note. Group elements that refer to the same concept closely together, and make sure that the spacing between them is consistent. Here is an example of how to better align elements on a page, especially for multi-panel figures. Draw a box around each step and rearrange them so that they align properly and the spacing between them is consistent.

During a user research call, various topics and methods were discussed, such as how to evaluate user feedback and compiling user research methods into a document. The importance of understanding user needs was also discussed, and a follow-up call was scheduled to further discuss user feedback. It was noted that quite a few new members had joined the room, including Francesca and Cindy who are on the BioRender team. A poll was released earlier, and although only a small fraction of the room responded, it was a balanced cross-section of those who had used BioRender and those who hadn't, as well as immunologists, molecular biologists, and others. Many attendees collaborate or receive feedback on their figures from fellow colleagues on their immediate team, and many have to make presentations for upper management, the management team, colleagues from the communications team, or others.

Here is an example of a quick before and after that was done in a live figure makeover. The basic science was there, but the flow of information was a little hard to follow. The image was turned into a Z formation, which was very helpful in cleaning up the story and making it much easier to follow. The principle of proximity was also used to group elements that referred to the same concept closely together.

They're better aligned, and that really cleans up the image right away. Sometimes it's literally just nudging it a little bit and leaving space in between the sections, so again, that's a principle of proximity. Here's another example to drive the message home. We took this up further and used sort of the idea of hierarchy of text and font and typography. So, you know, bolding the subheadings, throwing in numbers one, two, three, four, and then again nudging everything in place so that there's those nice visual hallways in between each step, and you get a nice separation of concepts per step. Okay, so these might again feel like very basic principles, but again, just getting our feet wet and sort of diving into making a complex figure as a pathway.

Tip number two: I'm going to go through a little bit of theory, and then three and four, we're actually going to dive into the application and do a bit of a workshop making the figure alive. A little bit of background - I don't know how many of you have ever taken a design course or learned about color theory, but there are basically three dimensions to color. You don't have to memorize this, just wanted to get it out there so you understand in case I use some of these terms. Hue is just another fancy name for the name of a color, so red is a hue, green is a hue, yellow is a hue, and each hue or color can have a value, which is basically just saying the lightness or darkness of the color. I would say this is the number one important thing to creating figures that colorblind audiences can see, to creating focus in an image. Chroma is the third dimension. If you think of it as an XYZ access, chroma is basically the saturation of a color.

Here's an example of such a pathway that we were talking about earlier that is actually created by somebody, a user in BioRender,, who has submitted and has been published in our template library. But wanted to showcase that even a figure like this, your eye probably went to this TGF beta first as well as to the phosphates because it has the highest level of saturation and contrast, and that's just where our eyes are going to go. Similar to how bees are attracted to the highest contrast area, which is the flower, the same goes for a pathway like this. You can think of it as the flower of your still life image or your pathway. If I were to now change the focus, change the saturation and value to a different part of the figure, your eye is going to go there first. So I bet you your eye went to the DNA or inside the nucleus in the second image in figure two, just simply by changing the color, the saturation level, and the value. So it's slightly darker, it's higher contrast, and it's a brighter color. So you have a lot of fine-tuned control over where you want your audience or your viewers to look first. Maybe that's the focus of your research, and you want them to make sure not to miss that part in a complex figure as a pathway.

Tip number three: Use opacity sparingly. If you're using too much saturation and contrast all over the page, it's like taking a highlighter and just highlighting the entire paragraph of a book or an entire page. You really want to use this sparingly, almost like a very powerful tool just in the right areas, and really for the most part, you can subdue everything to almost grayscale, just pastel or a softer color. You don't need to go in with a highlighter again and highlight every single protein. I just wanted to show you how powerful that is in focusing your viewer's attention to certain areas.

Are these three figures the exact same, just changing color? Okay, and one more point to touch on here, not necessarily a pathway per se. I'm sure you've made figures like this in the past. One challenge when making cell to cell figures is that the nuclei naturally stain darker, so I always see scientists struggling to create diagrams that have nuclei or something happening in the nuclei, and then it ends up getting really washed out because the nuclei are so dark. It's hard to overlay elements on top or layer them without it getting lost. One trick we like to use in industry is to convert figures to grayscale just to see where that contrast becomes an issue. Where there are really high value areas or low value areas, you can see here that the double-stranded DNA here actually looks single-stranded when I turn it to black and white. It just becomes very apparent that there are contrast issues when you convert it to grayscale, whereas in the color version, it's a bit deceiving because you think that just because it's a different color, it is able to be registered by the naked eye. And obviously, the label here, apoptosis, is disappearing. So what you'd want to do is bump up the contrast of those labels, and this is actually where we used opacity and played with the transparency to make the background elements a bit lighter. So basically, we took this T cell, and we decreased the opacity of it. So I think this is at 100 percent, and this one's at about maybe 80 transparency, which gives it a lighter color. It changes the color completely, and you have fine-tuned control when you change the opacity. You have to be careful if your background is really dark or something like that because obviously, that will start to show through in the cell. But if you don't, and we do recommend using a white background as much as possible, transparency is a very powerful tool. So, I think that drives a message about contrast, and what I love to do is touch on the last two tips here in the application itself. The last two tips were zooming in to show the correct scale, again to orient your viewer, and then four is the proper use of lines and arrows or at least the consistent use of lines and arrows. That's very, very important for telling your story correctly.

I'm going to go back into BioRender here, and what I can also do is pause for a second if there are any questions in the chat. It looks like there were a couple that may have been answered. Hi Shiz, one question that we had was how we can convert to grayscale in BioRender. If you could show that option, I think this is super helpful for following your tip there. Yeah, absolutely. Why don't I just reopen the figure here that I made just now? There's a couple of shortcuts here. There's this little black and white symbol, and actually, this little question mark that means there's a little tutorial that goes along with this feature. So if I click this, it's going to show me not only how to do it but also just the value or I'm going to use value for different reasons, the importance of using grayscale. So, I can click this button, and it's going to temporarily remove color on my canvas if you saw that. So there's the color version, and then here's a black and white version. There's this little button here. I can also just go to view-on canvas and grayscale. If I click that same thing, the cool thing is I can actually still continue to go about my drawing as if I was in color mode because it also allows me to see what is the actual true value of a color if I live in a world of black and white. So, you can see it changing to a different shade of gray. So, it's a really powerful habit to get into to think in terms of value and contrast. If you were to pick this color, I don't really know what it looks like in color, but I know that if my audience was colorblind, they could see it, and it's got enough contrast for even the average viewer to see. That's the convert to grayscale.

Just to get us warmed up in the app here, I wanted to go through six different ways to use arrows and lines. This is a really fast tutorial that I like to run everyone through just to cover the basics. If you've already seen this, that's great. This will be a nice refresher. One way to use lines and arrows is, of course, when you draw perfectly circular arrows to create kind of a feedback loop or maybe a cyclical pathway. We have this really cool circular arrow option in BioRender, so if you go into insert line circular arrow, all of these options are perfect circles. So, they don't behave like our other arrows, and you can't make straight arrows from a circular one. In fact, it looks like there's another little tutorial here that I could watch. I can cut the arrow into many pieces. I can change the length of that circular arrow, and it'll follow a perfect 360 arc. So, that's really cool. I'm going to close up that tutorial and I'm going to use this now. This figure that you're seeing here, we can probably upload as a template if you'd like to play around with it. This is just a figure that I've made using BioRender icons. So, I've kind of hacked BioRender to look like this worksheet just so we can show you the arrows option. So, here it is, a perfectly circular arrow. So, you don't have to do any guessing as far as how to make one, and I'm going to chop it in the middle here, so you can do that as well. I love this tool for showing circular motion activation. We can keep it as a pointed arrow, I think for inhibition; I'll use the flattened arrowhead. This one here, there we go. And maybe I'll even take it a step further and color code it so that, again, using color as a way to show antagonistic behaviors, look at that. And my canvas dimmed a little bit, and that's because I'm in what's called a grouped icon. It's kind of like an isolated world where, if I were to double click out of this now, this is going to move as one piece, whereas if I double click to the edit group, I can edit these objects, but I can't really select the things behind it. That's intentional. So, I'm just going to undo it.

That's using circular arrows, how to label things. This could come into effect when making pathways, when you need to label certain things and then differentiate those label lines from movement arrows. That's really important because sometimes what we see is the use of an arrow to show movement but also show it for the purposes of labeling. So, if you use something like this, it looks like maybe the vesicles are entering the cell as opposed to labeling it. That's why the consistent use of arrows and lines and label lines are very important. For labels, in particular, if you open up the most complex thing to label, like a neuroanatomy textbook, we always use lines with this little dot at the end. Even better is a line with a dot at the end and a kink in the line and here's why. So here's a vesicle and that dot at the end is pretty aggressive. I'm going to maybe decrease the size of that a little bit. You can change the size of that circle. There we go. Then I'm going to alt-drag just to copy. I think for a PC it's control-drag or maybe option-drag or alt-drag. Let me open up the cheat sheets here. If anyone is a PC user, maybe you can shout it out. Oh, duplicate. So, it's an option-drag for me because I’m on a Mac. So, it must be alt-drag for PCs. In any case, you can do that and very quickly copy as you're drawing. See how I've got that hinge in the elbow? It kind of just frees up that end to point to anything I like. Of course, avoid crisscrossing label lines as well and signaling pathway lines. Those get really confusing.

Let's just keep things neat and tidy. The contrast is getting a little dark in that area, but I think we're still doing okay. Moving on to the third kind of line is sectioning using dotted lines. Now, this kind of reminds me of using those paper doll cutouts as a kid. Dotted lines are great for things like cut lines for a pathway in particular, showing things that maybe did happen, or are going to happen, or maybe are less likely to happen than other events. That's a really effective way to use a dotted line. I wouldn't go too crazy with using dotted lines for every single part of your pathway because, again, it can get a little bit busy. These are all kinds of pro tips. So, I can't imagine using this for a lot of reasons, but if you want to get fancy, this is a cool way to show that.

If I were to zoom out, it kind of gives it that Nike Swoosh kind of moving through space, kind of an illusion. Okay, so we've just with these six different types of Arrow types, you can represent a lot of different concepts. I think that was enough review on lines. I'm going to go back to my folder here, and the nice thing about BioRender is it does auto-save. So, if anything happens to your browser, say Zoom takes up too much of my internet and it crashes my Chrome browser, I can actually go back in time using version history on any figure that I make. If I go back to say 2:33 PM, it's going to show the image that I started with. And then you can recover really any version of the figure that you're making. Just so you know that that's there as an option.

Great. And uh any new questions? I think we're doing okay here. Awesome. And then one thing I'll note is in making pathways, probably can't create one that's going to suit everyone's needs today, but um one elastic that I do want to leave with is the notion of creating softer curves and lines to your figure. So, you can see here we've got a pretty good start for a relatively complex looking image, a pathway diagram, or signaling pathway in an environment. You can actually change the line type from being one of those perfect elbow lines to a curve smooth line. It looks like this is already doing so.

So, every time you see this little line here that has it's hard to see at this scale, hopefully, you can see it. It's different from the corner node. It's got this kind of donut color to it, a white outline, the dark center. This actually will smooth out your curves. So, this is a nice way to make it look a little bit more waterfall and take off those hard edges of your pathway. I'd recommend that too if you want to make it a little more friendly. I think this will actually smooth out both edges, and this is a little nitpicky. You know, I think it'll just take your figure from looking 80% finished to maybe 100% once you start implementing these few options. And then if you haven't already noticed, within BioRender, we actually have taken some of that into consideration as far as the contrast and value options. These are all kinds of pro tips.

So, if I were to remove this, and I insert a shape with text in it, it's actually the exact same as if you were going to go down here and create a protein. Why don't I do that anyway just for the sake of the demo? So label, and I can type in "TGF beta." Beta is a Greek letter, so I'm going to come up here and insert a symbol. This is really nice because now I don't have to go down to Google and type in beta. Copy and paste. Actually, here's the drop-down, so you've got all the most common Greek letters. We even got these kinds of up and down arrows if you're showing increase or decrease, which is again very common in signaling pathways. You can go ahead and use these. But I just need the beta symbol, so I'm going to get that and delete these other letters or symbols, shrink it down a bit. I could change this into a pill shape if I'm talking about maybe a specific protein, so that's really nice as well.

So, just to be consistent with the top row, I'm going to make it a slightly curved object, and I like the color schema of the red background and the white text. You can see here the first two rows of our options is a dark letter font against a light background that's very intentional for us, and then the opposite is true, whereas a dark background and a light font. And this is important again for keeping that contrast. You want to kind of "quote unquote" foolproof so that you don't make any mistakes in your contrast when making signaling pathways. So here's that red option. I think whoever created this template did use one of our templated colors, so now it's matching perfectly. So that's a really nice way to start to include proteins and molecules into your figure. Again, I can option or alt-drag to create multiple stacks of proteins. Just makes it really easy, and I think the magic really is in kind of these pre-selected pre-vetted colors so that no matter what color you use, you won't have a risk of clashing, and that's another concept altogether is how not to clash your colors. I think that's a bit of an advanced workshop we should run, but in any case, using our preset colors for shapes, I think, is a good start to knowing that you're not going to have clashing colors or low contrast images.

Okay, and let's see, I'm going to hit preview just to see what that looks like. It's very nice and centered. The flow of information is quite nice. It's coming from up to down, and then sometimes we can't get away with doing two compositions, so it's up to down and then kind of left to right at the bottom, but I think, in general, it follows a pretty nice compositional flow.

All right, so I think in the last few minutes here, I think I'm just going to go ahead and show you how to make a figure from scratch, just to show you that it's not intimidating at all to start in BioRender. I'm going to set the foundation for my figure, which is maybe just a membrane. Again, some of you have used BioRender many times already, so this will be review, but I like to use the phospholipid bilayer whenever I can. Sometimes, it's not necessary, though, but we've got it pre-made here. This is an icon, which is kind of a flattened object. I can change the cytosol color, but I can't really change the individual phospholipid heads. If you do want to change the phospholipid heads, I'd recommend using our brushes option. It's represented here with this little blue brush logo. So, if I were to drag out this one that I think looks pretty similar to the icon version I dragged out, and I drag the end to the end of the canvas, this is actually a bit more flexible, and I can change the shape of this if I need. Again, going to go ahead and make it a darker color. I think it looks good. Kind of looking nonsensical, though, but that's okay.

Nucleus - just grab this one. First thing I see, the first one I see - hang it off the edge, by the way. Everything off the edge is not going to export, so you can use that as kind of like your workspace. TLR since I noticed that many of you are immunologists in the room, and you can use these simplified proteins sometimes, and we're still catching up here to these kind of canonical pathways, but sometimes we'll have groups or clusters of proteins already made that you can just drag out onto your canvas. And again, this is a grouped icon, so if I double-click it, I'm going to get into a different mode where I can again change the color of that pill shape, even change the label itself.

Already looking really nice, almost naturesque, and finally, again, this is going to be a little nonsensical, but maybe I'll add some arrows just to show that things are moving through space. Oops. I'm going to alt-drag to show that movement. Alt-drag again. Maybe then something happens here. Maybe it enters the nucleus at this stage, and then something happens. Copying and pasting here. Something happens later where it then exits the cell. Not really sure here, but just laying down the foundation.

Okay, and maybe I'll throw in some DNA just to finish off this figure. Lots of ways to approach DNA illustration. You can use, again, our flattened icons that are just beautifully rendered 2D vector shapes like this. I can change the color a little bit in situ. If you need a little more flexibility, we do have a DNA brush, similar to the membrane. For some reason, if your story is such that it needs to show a bit of a more complex shape, you're more than welcome to use our DNA brush. For this purpose, though, I'm just going to use this for now, and then protein. I love these kinds of generic protein shapes. I'm going to go ahead and use that. Anyway, a very, very simplified pathway here. Sure can get much more complicated, but just wanted to show the basics of how to get started. If I hit preview, there we go. And go back to my gallery. All right, so we covered a lot of concepts there, and I know we're coming up on time, so maybe I'll pause, see if I can address any questions that maybe weren't addressed.

There's actually a question from the audience asking what's the best way to visualize zooming in from a tissue level to a cell level to a protein level on the canvas. That's a great question. One thing that I would recommend is the idea of using kind of circle crops. Here's a simplified version: we take this from Anatomy drawings and that is using this kind of Zoom call out. This is a simple way to show zoom-ins of something that's quite complicated. What we did was create a circle crop of this mouse structure and put it around a circle. In the case of Pathways however, here's another example of showing the inside of one of these tiny wells and then showing that sense of scale in a really small area actually. So that's one way to do it. Another way we like to do it in cellular Pathways is to just draw a box around two shapes. You can shrink this down for example and remove a lot of the extraneous objects and keep it very simplified. If I were to be concentrating on a certain area, say that was my focal point of my story, it's obviously quite small and difficult to see at that scale, but you want to orient your viewer to say hey this is a cancer cell and a T Cell interacting. So what I do is make a little, sometimes they call it like a surgical window just kind of an orientation of hey this is the part of the body that I'm talking about and then of course zooming into creating that close-up but at a larger scale. I hope that's helpful. I would definitely say that that's one of the more popular ways to do it.

This becomes really tricky when you're showing, say, a virus interacting with a cell and then interacting with another cell so you get a lot of these kinds of changes of scale. If you can get away with showing something like this, that's great. Sometimes you could even turn the cell around to create that kind of environment. Of course, everything that hangs off the edge of your canvas is not visible so that ends up being your aerial focus. And of course, color matches to the thing that you're zooming into, whether it's blue and orange and then you don't get the kind of weird enormous protein on a tiny cell surface kind of thing. Preview there we go.

Sorry, that was a bit rushed but that would be one way I'd approach if you have multiple interacting cells and of course, you can multiply this to use against many other interacting cells as well. Thank you for the really great questions by the way and for those that are wondering how I'm panning around here, I am pressing spacebar to move things around and if you don't have enough space you can always close up your library to add more space in your canvas but just so you know that that's there's an option. We're just coming up to three o'clock or wherever you are in the world it's three o'clock our time in Toronto. Thank you so much for sticking with us to the very end. Hopefully, you learned a thing or two and we really appreciate you joining us today.