Questions about the circulatory system in the brain

Stall Catchers The Science
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How many different types of blood vessels are there in the brain? I know that there are arteries, veins, and capillaries. I know that the arteries take blood away from the heart to the organs, and the veins take blood back to the heart, and the capillaries are where oxygen is exchanged for carbon dioxide in the body’s cells, but I really don’t know too much more than that. Are all of the blood vessels that we are seeing in the movies capillaries?

In some of the movies, we can see large bands of blood vessels that seem to be all tangled together. However, in most of the movies, the blood vessels are more or less distinct, and separated from one another. Do these two different groupings of blood vessels serve different purposes?

I feel kind of silly asking this next question, but I guess I’ll ask it anyway. When I look at pictures of the brain, I’m used to seeing neurons, axons, and dendrites, as well as glial cells, but we don’t see any of those things in these movies. Where are the neurons, and why don’t we see them? My first thought was that the blood vessels must be on the top layer of the brain, with the neurons below them, so we don’t see the neurons because they are located in a much deeper level of the brain. The problem with this however is that there are neurons located deep within the brain, and there has to be some way for the blood vessels to reach them, so there really should be blood vessels throughout the brain, and not just on the surface, so I don’t understand why we don’t see them in the movies.

I really feel like I should know what capillaries are, and how they are different from arteries, but upon reflection, I really don’t. I always kind of thought that the capillaries were just a specialized type of artery, but now I’m starting to think that arteries, and capillaries are actually completely different types of blood vessels. How are capillaries different from arteries?

From time to time, the expert will say that a blood vessel has an unusual morphology. I’m not exactly clear as to what the term morphology means as it relates to blood vessels. Does the term morphology only refer to the shape of the blood vessel, or does morphology refer to something more than simply the shape of the blood vessel?

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Hi Mike, Lindsay here (from the Cornell biomedical engineering lab).

How many different types of blood vessels are there in the brain?
Your understanding of the blood vessels in the brain is more than sufficient; we generally only talk broadly about arteries→ capillaries→ veins. I know you’re an academic so below is an NCBI article that goes into greater detail;

Are all of the blood vessels that we are seeing in the movies capillaries?
All of the outlined vessels are capillaries, but you do sometimes see the larger vessel types in the videos. These vessels are far larger than a single cell in diameter and so they don’t exhibit the same stalling phenomenon we are interested in. (Keep reading to make sense of how bigger vessels make it into the movies at all)

How are capillaries different from arteries? In some of the movies, we can see large bands of blood vessels that seem to be all tangled together. However, in most of the movies, the blood vessels are more or less distinct, and separated from one another. Do these two different groupings of blood vessels serve different purposes?
Note the illustration from Mayfield Clinic—it is good to start thinking about how capillaries are a kind of intermediary between veins and arteries. The middle capillary beds are where the exchange you mentioned occurs. There is certainly variation in the complexity of these capillary beds; some capillaries may stray farther from the main beds making them appear more distinct, while some remain tangled. As far as I know, the function of exchange is similar, the variation is just the vasculature’s attempt to service more volume.

Where are the neurons, and why don’t we see them?
Not a silly question at all in fact I’m glad you asked, the answer is pretty cool. All of those brain cells you mentioned are there, we just aren’t seeing them because of how we image. The imaging modality used to create the movies is called “in vivo two photon excited fluorescence microscopy.” Breaking down this crazy name may answer your question:
“two photon excited fluorescence” – the microscope employs a 2-photon laser to selectively excite fluorescent dyes we have artificially added. In other words, the microscope only shows us what we want to see. In the case of blood flow imaging, we add a fluorescent dye that labels all of the plasma (liquid part of the blood). As the plasma is the only thing that carries a fluorescent dye excitable by the laser, it is the only thing that emits light! So within the movies you are watching, the other tissues are all there, we just don’t see them. The microscope starts at the top of the brain, above the dura (a protective covering) and images successively deeper areas, the big vessels that you see in the beginning are in the dura, and the occasional big ones you see deeper are servicing the capillaries.
“in vivo” of course means the mice are alive, blood pumping, breathing etc during the imaging session. So as the focus of the microscope moves deeper and deeper from the top of the brain, the images taken are moving in both time and space.

In sum- we don’t see the other stuff because we didn’t put in a dye for the laser to interact with, so they don’t really emit any light! It is a strange concept to get used to that light exists out of our visible spectrum and behaves differently/ illuminates different things… the first time I used fluorescence I thought of the magic markers I had when I was a kid that only became visible with a blacklight! Similar idea as far as the light thing goes. (When the laser is on in the lab during an imaging session we can’t even see it, we have to use a special telescope kinda thing to see where the beam is!)

Does the term morphology only refer to the shape of the blood vessel, or does morphology refer to something more than simply the shape of the blood vessel?
We mostly refer to the shape, sometimes the path. The vessels for the most part display the same shape and kinds of paths, but we occasionally stumble upon one that looks like it took a sharp turn or branched more than normal—variations that we need to be careful with as they sometimes confuse the computer’s total vessel count.

Please ask any clarifying questions and don’t hesitate to be honest if I just confused you more!

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The fluorescent dye explains the question of why the plasma is emitting light. Previously, I didn’t understand why the plasma would be glowing. Why does the florescent dye not enter the other cells through the capillaries? Is that because the molecules are too large to pass through the membrane? What would we see if no dye were present? Would everything just be completely black?

I’m having difficulty understanding the concept of depth with relation to the movies. As we move deeper down to successive layers in the brain, is that movement an actual physical movement, or just an apparent motion caused by magnification? I think I understand now a little better why it’s so difficult to make out what we are looking at in the movies because we are looking at a two dimensional representation of a three dimensional space. It’s still very difficult for me to understand exactly what I’m looking at when I watch the movies. For example, I see large glowing circles, and also smaller glowing circles in the background that look like stars. In a three dimensional space, we interpret larger objects as being closer to us, and smaller objects as being farther away from us. Since we are looking at a two dimensional representation of an image on the computer screen for example, how do we know that the larger blood vessels are actually larger, and just not closer to us, and the smaller blood vessels are not really smaller, but just farther away from us?

When we look at a two dimensional representation of an image, there’s really no way to gauge distance. When you are actually doing the filming of the movies, do you see a three dimensional image, or the same two dimensional image that we see when we watch the movies on the computer screen? It just strikes me that without being able to look at the images with both eyes, as we can in the real world, it would be almost impossible to judge whether the blood vessel we are looking at is the target blood vessel that we are supposed to be annotating, or really the one behind it because we have no way to judge depth.

I tried the the mouse neuron identification game once or twice, and I’m not very good at it, but one thing that struck me about that game, is that they do provide you with the three dimensional representation of the neuron that you are mapping in the game. I think that if it would be possible for us to have a three-dimensional representation of the blood vessel that we are supposed to be annotating, it would make it much easier for us to see where one blood vessel ends, and another one begins. However, I do realize that at this point, a three-dimensional representation of the blood vessel would probably be too expensive, and difficult to create, but I wonder how they did it for the mouse neuron game called Eye Wire.

One other thing I was wondering is why it’s not possible to create a computer program that can catch stalls faster, and more accurately than a human can? It would seem that one could create a computer program that could recognize stalls much more accurately than a human being, but I guess it must not be possible because if it were possible someone would have already done it. I know that some computer programs can even recognize faces, so why can’t a computer program recognize the difference between a blood vessel that is stalled, and a blood vessel that is flowing?

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I’ll see if I can explain more about the imaging. The dye we use is attached to a large molecule so it does not penetrate cell membranes, so it stays in the liquid part of the blood without going into the tissues or into the red blood cells. The red blood cells are black gaps in the dye because the dye flows around the cells. As you have seen, there are also bright spots, sometimes quite large, through out the tissue. This is because there are some naturally occurring molecules that are also quite fluorescent. Some of these are associated with aggregates of proteins the appear in aging and in injury. To be honest, we don’t know exactly what are those molecules. So when there is no dye injection, you can still see occasional dots and “splats” of brightness.

Each frame of the movie is like a photograph of a slice in the brain. If the brain were like a building of many floors, each frame of the movie is like a map or a flour plan of each floor in the building. In this case each floor of the building is only 1 micrometer tall. The perspective and scale on each image is the same and all you see in 1 image is just what is on that plane. The large and small glowing circles and are all in the same plane, so size in the image does not tell you the distance.

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I’m sure it will be possible to in the future to do this. In the past when we have tried, we have not been able to get to the accuracy to match the human. Both humans and computers have little difficulty with the easy, clear images. However, in noisy images, or data with motion artifact (from the mouse moving or the microscope being bumped), computer algorithms have not done very well. One thing to note is that to develop a computer program to do this, you need a large number of correctly scored examples. So we would use the dataset that is now being scored by human StallCatchers to validate any algorithm. Machine learning algorithms also need large, validated data sets for training. So any future algorithm would likely rely on the data being analyzed now by you guys.

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Okay, thank you! So the individual black spots are the blood cells, and the white glowing light is the plasma. However, there are at least two other cases that I’ve seen, one in which the blood vessels are completely black, and filled in, and one in which the blood cells look like long black streaks instead of spots. When the blood vessel appears completely black, does that mean that the blood is flowing normally except that there is no plasma present? I’m really not sure how there could be blood cells without plasma, but that’s what it looks like.

What’s going on in the case where the blood cells look like long streaks instead of spots? Sometimes the long black streaks almost look like they are pointed at one end, and that is often a place where a stall has been annotated.

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The streaked appearance from comes from the way the images are formed. The images are taken one pixel at a time. The focus of the laser that excites the fluorescence is moved from point to point in at raster pattern from side to side going from the top to the bottom of the frame. It takes about ~1/3 of a second to scan the entire image area. If a red blood cells is moving quickly, it only shows up in a few lines in the frame before it is gone resulting in a streak. If it is moving slowly compared the speed of the laser scanning, then you can see the whole cell.

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If the mouse is anesthetized, why would the blood be flowing faster at some times than at other times? If the mouse is unconscious, wouldn’t the blood be flowing at about the same speed all the time? I guess it might be the rate of scanning that is changing, and not the rate of blood flow. Is it the speed of scanning that creates the difference, or is the rate of blood flow actually faster at some times than at others?

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Hi there, I’ll try and chime in! The average rate of blood flow depends on various differences in vessel size, location in the body, blood pressure, stage of the heartbeat etc. AND there can be some variation at the cell level (not an average, more what you are talking about). Parts of this remain an open puzzle but there are some neat theories already published:

So whether the cell appears to be a streak or not really is the speed of the cell itself (considering the scanning speed remains constant and is sometimes slower than what’s needed to capture the exact shape as the cell moves).

You referred to vessels appearing completely black in the original question as well. Keep in mind that we are moving in both time and space, so when you are scrolling and the vessel fades out, you are passing the cross section of that capillary and moving into some space outside of the vessel. If the focus of the laser is no longer within the capillary there will be no signal. Am I interpreting that question correctly?

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What I’m referring to in saying that sometimes the vessel is completely black is that sometimes the vessel remains completely black throughout the entire movie, which is to say that as I scroll through the movie, I don’t see anything at all except the same continuous black field from one end of the slider to the other. So I guess my question is, when I don’t see anything except black, or gray, no streaks, no spots, no points of light, and only the slightest bit of movement from one frame to the next, what is going on there? Why is it completely black?

Also, sometimes I’ll see something that looks like a stall, but as I move the slider, a large amount of florescence seems to pass through the vessel, yet the expert still scores the vessel as being stalled even though florescence definitely passes through the vessel later in the movie. I always associate florescence with flowing plasma, but sometimes a large amount of florescence doesn’t always indicate a flowing blood vessel. Why does the florescence become so very strong at some points even though the blood vessel is stalled?

As I said in the original question, I believe that in the Eye Wire game, they display a three dimensional image of the cross-section along with a two dimensional image of the area that is being traversed. If there is some way that we could have a three dimensional image of the blood vessel in the stall catcher game, that would make it much more clear as to what is actually going on, although I’m not so sure how much it would help in identifying stalls.

One other thought occurred to me. Why does each movie not have a number, or a code associated with it? If each movie had a number associated with it, then if someone has a question about a movie, he or she could just refer to the number of the movie, and you would know which movie is being asked about. I assume that each movie must have some kind of a number, or code associated with it because otherwise you would have a really difficult time keeping them all straight, and organized, and you need to have some way of referring to them. On the other hand, the thought did occur to me that you can’t really show the numbers associated with the movies because if you did, that would be too much of a memory aid for the participants, allowing them to definitely remember whether or not they saw a movie before, and that could lead to presentation effects. However, it would still be nice if there were some way for players to be able to refer to the movies if they have questions about them.

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So I guess my question is, when I don’t see anything except black, or gray, no streaks, no spots, no points of light, and only the slightest bit of movement from one frame to the next, what is going on there? Why is it completely black?

That is simply volume that doesn’t contain any blood vessels! The bits of movement could be autofluorescence from other tissues that aren’t stained with the dye, but if a volume (a few frames) remains black it’s just that there was no dye in that area, indicating no vessels/ associated plasma.

Also, sometimes I’ll see something that looks like a stall, but as I move the slider, a large amount of florescence seems to pass through the vessel, yet the expert still scores the vessel as being stalled even though florescence definitely passes through the vessel later in the movie. I always associate florescence with flowing plasma, but sometimes a large amount of florescence doesn’t always indicate a flowing blood vessel. Why does the florescence become so very strong at some points even though the blood vessel is stalled?

To quote Nozomi, “The red blood cells are black gaps in the dye because the dye flows around the cells. As you have seen, there are also bright spots, sometimes quite large, through out the tissue. This is because there are some naturally occurring molecules that are also quite fluorescent. Some of these are associated with aggregates of proteins the appear in aging and in injury. To be honest, we don’t know exactly what are those molecules. So when there is no dye injection, you can still see occasional dots and “splats” of brightness.” So perhaps the expert is able to discern these artifacts by size, brightness, texture etc. noting that the bright white doesn’t quite look like the flowing plasma we see in our unstalled vessels (one of the reasons automation of this process is so difficult actually)

Maybe @pietro could share some thoughts on the rest? Thanks for the great questions Mike :slight_smile:

As I said in the original question, I believe that in the Eye Wire game, they display a three dimensional image of the cross-section along with a two dimensional image of the area that is being traversed. If there is some way that we could have a three dimensional image of the blood vessel in the stall catcher game, that would make it much more clear as to what is actually going on, although I’m not so sure how much it would help in identifying stalls.

One other thought occurred to me. Why does each movie not have a number, or a code associated with it? If each movie had a number associated with it, then if someone has a question about a movie, he or she could just refer to the number of the movie, and you would know which movie is being asked about. I assume that each movie must have some kind of a number, or code associated with it because otherwise you would have a really difficult time keeping them all straight, and organized, and you need to have some way of referring to them. On the other hand, the thought did occur to me that you can’t really show the numbers associated with the movies because if you did, that would be too much of a memory aid for the participants, allowing them to definitely remember whether or not they saw a movie before, and that could lead to presentation effects. However, it would still be nice if there were some way for players to be able to refer to the movies if they have questions about them.