Viv-Rolland

Leafing Nothing Out with Viv Rolland 001

 

 

[Music plays and text appears on a blue screen: Leafing nothing out, Image breakdown with CSIRO’s plant microimaging and biotechnology expert, Dr Vivien Rolland]

 

[Image changes to show a slide of a plant leaf under a microscope, and Vivien Rolland can be seen inset talking in the top left, and Summer Goodwin can be seen listening inset in the bottom left, and text appears: Vivien Rolland (Internal), Summer Goodwin (Internal)]

 

Vivien Rolland: So, what you’re looking at is a plant leaf, or a part, a tiny, tiny part of a plant leaf. It’s from the leaf of a tobacco species called nicotiana benthamiana. It’s not that important but it’s actually an Australian species and we use it because we can introduce the genes and proteins and either test their function, so what they’re doing or where they go within cells which is quite important for, for their function.

 

If you want to be cured you go to the doctor, like leaving your house to go to the pharmacy or the doctor will result in something different than if you go to the bakery, right. So, it’s not just about going out and doing something. It’s about where you go to be able to do the thing you’re trying to do right. So, it’s really important that a protein ends up in the right place to be doing the right task.

 

[Image shows the cursor pointing to the stomates on the microscope slide]

 

So, the different visuals, so this, these things here that look like lips, they’re called stomata, or stomates and they’re basically – OK, I don’t know if you can see the sort of the middle of the lips here but they’re, it’s actually a hole, and that’s where water and air can come in and out of the plant. And the plant can regulate this by closing it or opening it to let water either in or out. Right, so for example, if it’s water stressed, like if it’s really hot it would tend to close it to preserve water. Otherwise water evaporates right. But it also needs to have them open because it needs to get air inside because it needs the CO2, that carbon dioxide to be able to make energy, right. So it’s kind of a balance between letting air in and not losing too much water. So, it’s really important that’s, that’s regulated. So, that’s one thing and they’re a feature that you find on lots, and lots, and lots of plants, alright. And they have a similar shape. They vary a bit from species to species but they’re easily recognisable.

 

[Image shows the cursor pointing to the outline of a cell and Vivien can be seen talking inset at the top left, and Summer can be seen listening at the bottom left]

 

And so, the other things that you can see are sort of like, see these lines, they’re outlines of a cell. So, for example if you follow my mouse here.

 

Summer Goodwin: Yep,

 

Vivien Rolland: That is delineating a single cell. So, that’s one, so inside that is one plant cell right. And then you’ve got, here you’ve got another one, right. So, you’re looking at multiple cells within this space and the line itself is the cell wall which is, plants have a cell wall which is made of mostly cellulose and lignum and things like that. And it’s kind of a rigid structure which animals don’t have. So, when we have two cells touching each other as a human, there’s no cell wall in between them. That’s something that’s, that’s one of the differences between plants and animals. And that’s, that constrains the shape of the cells and their, their ability to grow of course, right. It’s kind of like a, think of it as a, as a box right. You sort of, if you want to grow you have to make the box bigger right before you can actually grow.

 

[Image continues to show Vivien talking inset on the top left, Summer listening inset on the bottom left, and the cursor can be seen pointing to the nucleus in the microscope slide of the leaf]

 

And then, so that’s, that’s one cell wall, and then you have these bright dots here. They’re the nuclei. So, the nucleus of each cell is where the DNA is.

 

[Image shows Summer inset at the bottom left agreeing with Vivien, and then Vivien continuing to talk inset in the top left, and the cursor continues to point to cells on the microscope slide]

 

Summer Goodwin: Oh right.

 

Vivien Rolland: Yeah. So, that’s why you have one here for this cell and you have another one here for this cell. You have another one for this smaller cell and there should be about, there should be just one per cell. And yeah, so that’s, that’s what you see. So, all of those…

 

[Image shows Summer inset at the bottom left talking, and then Vivien continuing to talk inset in the top left]

 

Summer Goodwin: I wasn’t expecting the cells to be all so, such different shapes. I kind of had this idea that cells would have some uniform structure but…

 

Vivien Rolland: No, so that’s a really…

 

Summer Goodwin: Beautiful.

 

Vivien Rolland: So I guess the first cells like at the very beginning of the development of a human, or a plant, or whatever, they don’t really have a specific, they don’t have an intricate shape like this but there’s this process called differentiation which is basically that you have… so in your body you have neurons, you have muscle cells, you have skin cells, and all of these are like, you have gut cells, they all have a different function. And to be able to have that different function they usually have a different shape, right, that kind of fits the purpose. The neurons have these long tubes called axons that they use to transfer information. A muscle cell doesn’t need that, it needs other things. A skin cell doesn’t need that, like skin cells need to be tightly connected so that you actually have a barrier right, that protects you from the environment. Whereas, neurons need to have these long extensions so they can connect to other cells, right.

 

So here these cells are intricately connected like this probably to, to form a tight barrier, right. It’s kind of a, so we call them jigsaw puzzle because they look like this. So, just underneath actually, so this is really interesting because the, we’re getting into like what the colours mean, right. There’s actually multiple layers that you see right now. So, the very surface of the, so imagine how do I do that?

 

[Image shows Vivien inset in the top left holding up a block of wood to demonstrate, Summer can be seen inset in the bottom left listening and agreeing, and the microscope slide can be seen]

 

OK, so imagine this is what we’re looking at from the top right. That’s the surface of the leaf but actually if you look at it the other way, you’ve got one surface on this side, and one surface on this side, and that’s the inside of the leaf, right.

 

Summer Goodwin: Yep.

 

Vivien Rolland: So, you’re actually looking from the top here but you’re looking at maybe this far down.

 

Summer Goodwin: Ah, I see.

 

Vivien Rolland: So, you’ve got multiple layers right. Yeah. So, the lips here, the lips, the nuclei and those lines here, they would be the first layer, right. That’s the barrier layer, the layer that protects the, the leaf from the outside.

 

[Image shows Vivien talking in the top left, Summer inset in the bottom left listening and agreeing, and the cursor pointing to cell layers on the microscope slide]

 

But then actually underneath it you have another layer and you can start to see, so this here, I don’t know if you can see this blob here, or this blob here. So, this is actually the cell layer that’s underneath it.

 

Summer Goodwin: Right.

 

Vivien Rolland. And they actually have a different shape.

 

[Image shows Vivien demonstrating with his hands epidermal cells inset in the top left, Summer listening and agreeing inset in the bottom left, and the microscope slide on the main screen]

 

They’re actually, if you think of the, the, surface of… so these cells are called epidermal cells like on our skin right, the epidermis, and then dermis. So, the epidermis is on the outside. So, they’re like this and then underneath you’ve got cells that would be like this, right. And so that’s why you start to see the tip of some of them popping through here, right. If I go deeper in this you’ll start to actually, you’ll stop seeing this, and you’ll see just these cells right.

 

Summer Goodwin: Oh, got you.

 

Vivien Rolland: So, there’s a lot in this image right. Now, let’s make it even more complicated.

 

Summer Goodwin: Yes please.

 

[Image shows Vivien talking inset in the top left, Summer listening and agreeing inset in the bottom left, and the cursor pointing to the strings on the cells on the microscope slide]

 

Vivien Rolland: So, these strings here, they go across the cells.

 

Summer Goodwin: OK.

 

Vivien Rolland: So, they’re actually part of the solution that makes the cell, right. They’re kind of liquid of the cell in which the proteins are and everything, right. And plant cells have this big kind of balloon in the middle of the cells that pushes everything against the cell walls. That’s why actually most of that looks empty because this is basically a big balloon that’s like blown out against the cell walls and it’s basically, it’s called a vacuole. It’s pretty much full of water and so that’s why like, you know, salad is actually full of water because actually nearly all of that is just water with some, some sugars, some minerals, some this and that. So much water in it, right. And that’s what actually keeps the shape of the cells is this sort of balloon that pushes on the outside of the cell wall. These things are just going across to be able to connect different parts of the cell, right. So, they’re sort of like going through the balloon without piercing it.

 

[Image shows Summer inset talking in the bottom left, Vivien inset in the top left listening, and the microscope slide can be seen on the main screen]

 

Summer Goodwin: OK, yeah. They’re kind of holding it all together.

 

Vivien Rolland: Sorry.

 

Summer Goodwin: No, I love it, to kind of hold it all together because it’s mostly water. So, do they act as kind of, like keeping the whole thing together?

 

[Image shows Vivien continuing to talk inset in the top left and demonstrating with his hands, Summer inset listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

Vivien Rolland: The water is enclosed in a balloon. So, there is a membrane on the outside right and, but it means that if you want to travel from here to here, you typically have to go all around, right. So, these strands here they basically travel through, and they’re like a highway, they allow some things to move across the cells in different ways. So, if you look, you’d have some here, some here, some here. So, they’re kind of these connecting tubes. Now, one last thing is what do the colours mean, right. So, if you looked at this with a normal microscope you’d just see green.

 

Summer Goodwin: OK, yep.

 

Vivien Rolland: Right, because that’s, if you grab a leaf, I don’t know if you’ve got plants around you but I can see behind right, it’s mostly green, sometimes a bit of white, but that’s green and that’s because of the chlorophyll.

 

[Image shows Vivien inset talking in the top left, Summer listening and agreeing inset in the bottom left, and the cursor pointing to the chlorophyll discs on the microscope slide]

 

So the chlorophyll is found in those tiny discs that are inside the cells. And particularly this one. Actually all these black areas here, that’s the negative imprint. That’s where there would be a, there would be an organ here full of chlorophyll. There would be another one here, there would be another one here. There would be another one here. So, they’re, they’re called chloroplasts and they basically capture the sunlight, use water, and CO2 and make energy right. So, if you look at a leaf normally, with a normal microscope or with your bare eye all you see is green. But there’s the thing that’s quite magical is that it is green because it absorbs, so, OK, so if you think of white light, so the light that we’ve got from sun or light you’ve got from your incandescent or LED, you know, light bulb just above you, it’s basically all colours of the rainbow.

 

Summer Goodwin: OK.

 

Vivien Rolland: So, it’s all colours of the rainbow, and the reason why we see rainbows in the sky is that, is after it rains, that light actually when it goes through the water that’s in the air it gets diffracted, so all the colours that are in that white light just get separated and you get to see them individually. So, you get purple, blue, green, yellow, orange, red, right. That’s the range we can see. Normally we don’t see it, it’s all together and it looks white. But when it goes through water because these colours have different properties they get separated and you actually see a rainbow. So, here what we’re doing is we’re actually utilising the fact that light is made of different colours and we pick say, we’re going to use blue light, and we’re going to use red light. And we shine only a blue light or red light onto this leaf. And the unique properties of what’s in different parts of the cells, and what’s a different part of the plant, react with this light and some part will light up, and some part will not. You know, it’s called fluorescence. So, some parts will absorb this energy, this light, get excited, but can’t stay excited, it needs to release this energy, and it releases this energy in the form of light. And that’s called fluorescence.

 

Summer Goodwin: Right.

 

[Image continues to show Vivien continuing to talk inset in the top left, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

Vivien Rolland: And so, for example here, different parts of the cell would react differently, right. Now the last, so that’s called fluorescence, [11.48] microscopy, it’s pretty intense but that’s just the gist of it. The last thing you need to know here is that there is, I have actually used a dye here.

 

Summer Goodwin: OK.

 

[Image shows Vivien talking inset in the top left, Summer listening in the bottom left, and the cursor pointing to the blue and black areas on the microscope slide]

 

Vivien Rolland: So, I’ve injected a dye and that’s why you see this sea of blue. It’s because do you remember how I said like these have pores, and air and water can come through?

 

Summer Goodwin: Mm hmm.

 

Vivien Rolland: That means that underneath, within that leaf, there’s actually an air space, right.

 

Summer Goodwin: OK.

 

Vivien Rolland: And I’ve filled most of this air space with this dye. And that’s what you see in blue here. And the parts that are in black are parts of the air space that haven’t been filled, OK.

 

Summer Goodwin: Yeah.

 

Vivien Rolland: Now what is really, and this is probably getting too complicated, but what is really cool here is that both the pink and the blue colours are due to the dye. And that’s because, so the dye has also gone into the nuclei. It’s also gone into the cell walls and all that but because this dye is special it changes properties with the pH. You’ve heard of the pH, it can go between acidic and alkaline, and water is neutral in the middle. So, lemon is acidic, coffee is acidic but water is pretty neutral, and then soda, baking soda, or bicarb is very basic.

 

Summer Goodwin: OK.

 

Vivien Rolland: Right, so, so what’s really special about the different parts of the plants is that their pH is different.

 

Summer Goodwin: Got you.

 

[Image continues to show Vivien talking inset in the top left, Summer listening and agreeing inset in the bottom left, and the cursor pointing to the cell walls on the microscope slide]

 

Vivien Rolland: And that’s for a number of reasons. For example for the cell walls to grow they need to be acidic. So, that’s why, but when it’s just in the air space like this it’s actually more basic. So, when it’s more basic, it’s blue, when it’s more acidic it’s pink, right.

 

Summer Goodwin: OK.

 

Vivien Rolland: So, it’s got these different parts.

 

[Image shows Summer talking inset in the bottom left, Vivien listening inset in the top left, and the microscope slide can be seen on the main screen]

 

Summer Goodwin: Sorry, basic is another word, basic is another word for alkaline?

 

[Image shows Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

Vivien Rolland: Yes, sorry yes. It’s more alkaline, let’s keep alkaline, yeah, yeah. So, it’s more alkaline. So, that’s what, so that’s what’s in this image, right.

 

Summer Goodwin: Right.

 

Vivien Rolland: And the reason, and it’s probably not what you want to put in the box, it’s way too complicated but this is just for you, like you know, to walk you through the different layers that are in this image.

 

[Image continues to show Vivien inset talking in the top left, Summer listening and agreeing in the bottom left, and the cursor pointing to the nucleus on the microscope slide]

 

Now, there’s another layer which is that normally you would not expect the dye to go into the nucleus because the dye can’t go through membranes. The reason why it’s going into the nucleus is probably because the membranes of the cell were broken. So, probably when I, either the cells were stressed or when I introduced the dye I must have broken some membranes and it’s gone inside the nucleus as well, right. So, so that’s what’s behind this image. So…

 

[Image shows Summer talking inset in the bottom left, Vivien listening inset in the top left and agreeing, and the microscope slide can be seen on the main screen]

 

Summer Goodwin: Wow. So, back to the alkaline and acidic. So, when you’ve put in the blue dye has it come up pink because it’s more acidic or more alkaline than the other parts of this cell?

 

Vivien Rolland: Yeah.

 

Summer Goodwin: Yeah.

 

[Image shows Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

Vivien Rolland: So, what it, so basically… Yeah so it’s, it’s, and that’s why I was using it but that was one of the very first, actually I think it was the first time I used it and so it was one of the first images I captured and I thought it was beautiful so I’ll take, and I was actually told by someone, “Oh you should put in an image for this competition”. And I thought, I’m just imaging that, maybe I’ll do something around it. You know, that was just serendipitous. This image per se can’t be used for science because it’s not really answering any question, you know, but it was just, we were experimenting with this dye and we thought, “Oh that looks really good”.

 

And so, the basic thing is like I’ve injected a dye in air space of plant leaves, and that dye has chemical properties that means that it reacts differently when it’s in an acidic or an alkaline environment and we can visualise this with these special lasers. And when it’s acidic it’ll do pink and if it’s more alkaline it’ll be blue.

 

[Image continues to show Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen changes to show a yellow and blue leaf]

 

So, what it is is you’re looking at plant leaves. You’re looking at mostly the epidermis. So, the very outer layer, right.

 

[Image shows the cursor pointing to the cells walls on the microscope slide]

 

And you can recognise the, the cell walls here. So, that’s really good.

 

[Image shows the cursor pointing to the highways on the plant leaf on the microscope slide]

 

As you said, this and this and this, and this, they’re all sort of highways that go through that vacuole, which is this balloon full of water, alright. And, and the difference, the reason why this looks drastically different is two things. One is we’re not using the dye. So, I haven’t filled the spaces or anything. Instead we’re tracking a protein that has a fluorescent tag. So, it’s basically, it’s got a tag attached to it, so whenever you can see the tag, you know that you’re looking at the protein because they’re attached to each other. Now, the, this protein is expressed, when we say expressed it means it localises, in that, what I was saying, that sort of fluid that fills the cells.

 

[Image continues to show Vivien talking inset in the top left demonstrating with his hands, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

So, imagine you’ve got the balloon. You’ve got the hard scale wall which is on the outside and between that there is this sort of soup. It has proteins, little organelles and things like that, right this is where the action takes place. And so, literally this protein is expressed in that soup. And so, that’s why those streams look actually quite big compared to the previous image because the previous image was just the dye who had sort of sneakily got in. So, you only had a little bit of it whereas here you should see a lot more of it because you’re actually highlighting, you’re basically making that soup glow. This is exactly what it is, you’re making that soup glow, right. Now why do we have these different colours, you were on to it. It is because I’ve taken what we call a stack, so same thing with the previous image.

 

[Image continues to show Vivien talking inset in the top left and demonstrating with a block of wood, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

I’ve taken actually, if you go back to that block right, I’ve taken an image here, an image here, an image here, an image here, an image here, an image here, an image here, an image here, see I like keep going down. And then we can actually put that together, right and that’s what I’ve done here except that I particularly, because we’re looking at just one protein, you know, one type of protein, if I don’t do anything it will be either green or yellow, or whatever I want it to be because of its colours. But here what I’ve done, is I’ve said, OK well, the uppermost planes, the ones that are the most on the outline would be let’s say yellow, and the ones most at the bottom would be blue, and between would be a gradient. So, what you’re seeing basically is kind of giving you an indication of depth. When you go from yellow to blue, you go, OK I can’t remember which way but you go from the surface to deeper or the opposite, right.

 

Summer Goodwin: Yeah, beautiful. Yep.

 

[Image continues to show Vivien talking inset in the top left and demonstrating with a block of wood, Summer listening and agreeing in the bottom left, and the microscope slide on the main screen]

 

Vivien Rolland: So, this in itself is not very useful. It just looks good. It’s not very useful, right. So, if I… if I was using this image for science I would probably pick a single, a single plane, a single part, and I’d just have one colour because it, you know, so I just did that because it looked great, and I think it looks like there’s a scary monster here. So, if you really want…

 

Summer Goodwin: Yeah, it does that too.

 

Vivien Rolland: Yeah, and the reason why you can’t see, OK so there’s a couple more things you can see if I tell you where they are.

 

Summer Goodwin: Yeah.

 

[Image continues to show Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the camera zooms in on the microscope slide and the cursor points to features]

 

Vivien Rolland: So, you can see how like you’ve got these little black things within that stream…

 

Summer Goodwin: OK.

 

Vivien Rolland: Can you see that, there’s a little black dot, there’s a little oblong thing, there’s a black line in the middle of that, and you can see some here as well. OK, so remember how I told you like it’s highlighting that soup, well that soup has little organelles in it, like mitochondria, things like this that make energy, chloroplast, so there’s probably chloroplast in here, or actually in here, and this could be mitochondria or other organelles. And so, these don’t glow. So, you just see them black and then around it is all glowing. So, that’s… that’s why you have those what I said like negative imprints in a way, you know. So, there’s one here.

 

[Image continues to show Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the camera zooms in on the microscope slide and the cursor points to features]

 

So, there’s probably a chloroplast that sits in here and then the soup around it is glowing and technically that’s why you can also see things like, it’s a bit hard because it’s a depth thing but the black line in between here is probably where the cell wall is. So, you’ve got the soup next to the cell wall on one cell, and then the soup next to the cell wall on the other cell, and they both glow but the cell wall itself doesn’t glow. So, you get a black line in the middle. So, that’s what you’re looking at here.

 

[Image shows Summer talking inset in the bottom left, Vivien listening inset in the top left, and the microscope slide can be seen on the main screen]

 

Summer Goodwin: And what about the red, because that…?

 

[Image continues to show Vivien talking inset in the top left demonstrating with his hands, Summer listening and agreeing in the bottom left, and the microscope slide can be seen centre screen]

 

Vivien Rolland: So, the red would just be another plane. So, the leaf will not be flat. It will not be like this. It’s probably a little bit like this so that when you start imaging the plane might just, the first plane might just cut this corner and then nothing else. So, maybe… maybe it starts at red rather than yellow. Maybe it starts at red and goes to blue.

 

Summer Goodwin: Oh, I see, yep.

 

Vivien Rolland: And then, and then the yellow is in the middle. So, it could be that this corner here is a little bit higher up and the first plane kind of cut through that and the rest was just black. There was nothing, right. And then as, and then as you go down you start to cut through the whole part of the leaf. So, yeah.

 

[Image shows Summer talking inset in the bottom left, Vivien listening inset in the top left, and the microscope slide can be seen moving up and down on the main screen]

 

Summer Goodwin: Yeah, beautiful. Oh that was so interesting.

 

[Image continues to show Vivien talking inset in the top left, Summer listening and agreeing in the bottom left, and the microscope slide can be seen centre screen]

 

Vivien Rolland: Yeah, so I mean this is like in way more detail than you need to tell people probably but this is more like, you know, for you, and out of interest. OK, so the first image we took it as part of an Honours project with one of my students.

 

Summer Goodwin: Yeah.

 

Vivien Rolland: And she was looking at how, how bacteria go inside roots to form the symbiosis that basically fixes nitrogen. So, you know, nitrogen is fertiliser basically, is a big problem in ag. There’s some species of crops that know how to fix nitrogen, to capture nitrogen from the environment and they do that by creating this interaction with bacteria that are able to do this. And the bacteria live inside the plant roots. And the plant root provides protection but also sugars and things for the, for the bacteria. So, it’s a symbiosis. They both benefit from it. And she was looking at how the, the establishment of this process, and how the cell walls are modified during this. And so that’s why she was looking at this dye, or she wanted to use this dye which turned out to be impossible to work with. It’s way too difficult. But to look at this dye to see changes in pH in the cell wall as they were reacting to these bacteria. And so in this case I was following a protein to make sure I could target it, send it to the right part of the plant so it could carry its function.

 

[Image changes to show the CSIRO logo on a white screen, and text appears: Australia’s National Science Agency]