 Good morning. Here we are at the Lamont Doherty Earth Observatory. This is the tree ring lab and my name is Arturo Pacheco. I'm a forest ecologist and I'm working here at the tree ring lab developing a technique that is called wood anatomy. This is part of like a broader science which is denrachronology. What we do here is to try to get really thin slices of wood samples so that we are able to see the anatomical features of it for different purposes. So some of these purposes can be the health of the tree, how old is the tree, how it is growing. It can also be used for for example in archaeology. It has been used to being able to date all the structures and also being able to tell what type of wood is a construction made of and all just that but we are able to tell the origin of that wood, I mean where it is coming from. Dendrochronology is the broader part of this science when we are using the tree rings to be able to put the information that the wood gives us on a time context. The classic approach is by using increment borders to get samples from the trees and by sanding these samples we are able to make the tree rings very clear and under a microscope we will be measuring every single tree ring. This will give us basic information of how good was the growth season of each year and that way we can also check on climate correlation so that we are able to see when the tree was growing really good so probably very warm years, it was also very rainy years depending on the site. Wood anatomy is kind of like a sub-part of dendrochronology where we are doing micro slides from the wood so we take our sample that we use in classical dendrochronology and with this type of machine that we call microtones we get very thin cuts of our samples and those cuts then we dye them so that we are able to see the cells so we go from a very macro level in dendrochronology into a much more microscopic level. Over there we will be able to see not just how big is the ring but we will be able to see all the cells within it and we can measure those cells so we can start getting information at an intra-annual level so we will be able to see what happened during that growing season and not just saying okay it was a good or a bad year. I'm going to show you what's the process the methodology and the protocols that we use to get these wood anatomy samples so from the very beginning we have this type of samples with a saw machine we just get a strip of wood that is what we are gonna need and this is what's gonna go into the microtome. The very first step that we have to take with the wood that we are gonna cut together where anatomical sections is to boil it. The classic and the standard way to do it around 10 minutes for softwoods and probably around half an hour for some of the hardest hardwoods that we have out there. Now I will show you the basic tools and instrument that we use to start our process of getting anatomical cuts from the wood so first of all this big machine that you see here is what we call a microtome in this case is an sliding microtome we will put our sample on this platform here and then we use very simple tools. Normally for us to during the cutting process we will just use a mix of water and alcohol 50% 50-50. The samples we will put them on a glass slide like this one where we will be able also to write the name or the code of our sample and we will help ourselves cutting with a little paintbrush like this one and also a couple of tweezers and with that we should be able with that and with a lot of practice and a little bit of talent I would say we will be able to get a very thin cuts from from this wood. How thin? Normally we use this part of the microtome is to graduate how thick is the cut that we are doing and normally we will be cutting the wood from 10 to 12 micrometer that's kind of like the thickness that we will need for this type of preparation. Okay I think we are there to make some nice cuts. Well now after we have got our cuts from the microtome we have to move to the hood because we are starting the coloring process of our samples. First step we will put some bleach in our samples because that will help then the color to stick on our tissues on the wood. After that we will wash out the bleach so I just take out the bleach with a bit of water. After that we will go with our main colorant that we use that is Saffronine and Astro Blue. These are two different colorants so basically the Saffronine is a red colorant that will attach itself to the lignin of the wood while the Astro Blue will attach itself to the cellulose of the wood. So once we have already had our samples around 10 minutes on Saffronine we will just start washing it with water to get the excess of the Saffronine out. As you see we will be able to see how the Saffronine just goes out of the sample. Once we get most of the excess of Saffronine out what I do is to use a small piece of paper tissue just to kind of like block our sample so that they will not be moving around and we start washing it with alcohol 50 percent because even though here it seems that we have already cleaned it you will see that the water will still bring out a lot of the colorant and we will have to do this for a couple of minutes so when we are here that we don't see that much pink color coming out of of the water we can pass to the next step which is the alcohol 99 percent when I just keep it like for a minute with the alcohol 99 percent so that it will kind of like go into the tissue and bring out the rest of the of the dye that we have so we just we will just keep on washing it. At this point we just bring it here over a paper towel and we prepare our aeokit which is our mounting material which is the one that will make our samples a permanent sample. First we take out the small tissue that we use to let's see we have to be really careful there we go we reposition our samples at this point they they are very sturdy so it's not a problem to touch them with your hands and here we have to dry it and this is kind of like the trickiest and the quickest part of of the process I do it once I do it twice and I already prepared the aeokit so in that case I put a couple of drops in each of the sides so that it will not start drying and coral in as you see it's already you can see it dry a little bit but the drops will maintain it in its position and here we have already enough mounting material to cover our glass slide so at this point we just need to bring it cover it and we will just leave it for a couple of minutes that the mounting material will spread all over the sample we want this to dry completely but under a little bit of pressure so that the tissue will always be as flat as possible so in my case I'm using this plastic also to to protect the surface because this mounting material can come out so I put my samples inside these little plastic backs here and I have the metallic surface and these magnets so basically once you have it there we just cover it with one of these long magnets and a second one just to be sure that is well pressed after we have finished getting our anatomical cuts and after one day of leaving them to dry we will be able to clean them a little bit first before coming here to the microscope stage we have to be sure that we don't have any type of specks from the mounting material or anything so we will be able to get a very clear image of it in this case what we are using here is a very simple microscope the one that you will find in most laboratories and over here we are able to see really well all the features in the wood for example in this case here we are looking at an angiosperm this is a softwood type of and what we can see is very clearly the vessels this is where the plant is transporting the water mostly and all the fibers that are the ones that are giving sustain to all the the tissue of the plant here I want to show you one of the applications of wood anatomy in our lab we are doing a lot of climatic correlations with our samples from tropical species so basically we need to measure ourselves and do correlations with the weather pattern and the climatic data that we have from that site so that we know how the tree is responding to let's say gears that have longer dry seasons or longer rainy seasons and to do that we use a software that is Roxas which is a software that will automatically measure all of the area and many other anatomical traits on the image in an automatic form and it will generate us a big data set that is the one that we are using to do this correlation okay so when we already have the image ready I want to show you just the automatic analysis that this program does so I have already set it for the species that we want it will just ask me to to find an area of interest so I will just tell them that to analyze me I square here compressing three rings here is and okay so here the program will start going through a lot of different filters it will start analyzing the whole image taking out like shadow like a small some cells that are not perfect so in this case I'm using a very small image because it will take a long time normally with the images that we are producing it can take over 40 minutes but that's because we are measuring millions and millions of cells here we are just measuring probably 500 cells or something like that and in well in this case just in two minutes since we started we were able to get the analysis of the image here you can see the cells that the program has captured if you are able you will see like these dark spots those are like the the rings and also vertically there are some dark spots also those are the rays that are areas that we do not want to measure so in this case we are just measuring really the cells that we are interested in which are mostly the vessel the biggest ones and some of the of the small fibers I can change this here into the image with with the real image and for you to be able to see clear ourselves we can do this and here we can see the results normally automatically we can design the the rings in this case I will just do it manually because it's a very small image so I can show you we normally visually can tell where the ring is positioned so it's just a matter of give these parameters because then the program will calculate all the data from for these cells and will position them on on the gears that we will tell the program in this case we collect them in 2017 so I can just put here 2017 so that it will give me it already give me the gears I have to update it and here you can see already the program has assigned the the gear of study 2017 16 and just one piece of the 15 and after that it's just a matter of calculating output and the program will give me a txt file and also if you want an excel file with all the different traits of anatomical traits that the this program is capable of measuring that is the area of each cell the diameter of each cell how they are grouped how far away are they from the limit of the ring so at the end we will have a lot of data to do our correlations and we will not be able to do this if it's if it's not for our ability to get very good cuts very good anatomical samples so that the program will be able to measure it so the quality of our sample is the most important step for the computer to understand the cells and give us the most accurate data for our objective on research in my background is forest ecology I have been working on the specific part of dendrochronology I started working first in in agricultural sciences and then I started liking a lot the ecological connections with the forest so I started moving into this part of forest ecology and within this area I learned about dendrochronology