A Technical Perspective in Modern Tree-ring Research – How to Overcome Dendroecological and Wood Anatomical Challenges

The overall goal of the following experiment is to generate well replicated and highly resolved wood. An anatomical data sets to study the effects of time and environmental factors on tree and shrub growth. This is achieved by using a non-Newtonian fluid to stabilize the plant cells during microtome sectioning, allowing a detailed microscopic analysis of the samples as a second step.

The micro sections are double stained to facilitate the differentiation of the lignified and un lignified cells, and then digital images are taken of the samples. Ultimately automated cell-based analyses of the plant growth and related time series analyses can be performed to allow the evaluation of the wood anatomical parameters of interest. The main advantage of this technique over existing methods like sending the sample score that with our techniques, the cells are not filled with sawdust, resulting in much clear quality samples for image analysis.

Further, adding a non Newton fluid to the cells during sectioning allows avoidance of the time consuming technique of embedding the sample in paraffin. The new microtomes that we'll see in the video were developed in our laboratory incorporation of central Lu Netti of the Shang Dubs and Zurich. They stand out due to a high stability of the guidance, which allows the cutting of big and dense specimens.

For core sampling of tree stems. Begin by placing a sharpened increment core perpendicular to the growing axis of the tree stem. Next turn in the core and use an extractor to remove the resulting core sample from the tree.

Label the core with a specific ID marked in soft pencil on the core surface, and repeat the procedure on the opposite side of the stem when all of the samples have been collected. Store the labeled cores in an appropriate container to protect them from damage during transport. Then once back in the lab, mount the core samples fiber direction, upright on wooden support beams with water resistant wood glue, and let them dry for micro core sampling.

First, use a chisel to remove a portion of the bark as necessary. Then place a special punching device perpendicular to the growing axis on the stem has just demonstrated, and use a hammer to penetrate the xylem of the stem to about two centimeters in depth. Turn the punching device to break the resulting micro core inside the tool.

Then remove the core from the stem and store it in a labeled micro centrifuge tube. To cut a plain surface on a core sample, begin by fixing the increment core of interest in the core holder of a newly developed core microtome for cutting plane surfaces on cores over their entire extent. Then lift the core until it slightly touches the blade, which is fixed on a ball bearing guidance over the extent of the core, and pull the blade to cut the first part of the top of the core.

Next, push the knife behind the core. Lift the sample holder about 10 microns and repeat the cutting procedure until approximately one third of the core's. Diameter is cut down resulting in a continuous plain surface.

For detailed image analysis of the anatomical structure of the samples, micro sections have to be prepared with another microtome type. First, fix the core sample of interest in the microtome holder of the microtome. Then position the microtome blade guided by the ball bearing guidance and pull the blade over the core.

Push back the blade to lift the sample by 20 to 30 microns. Then pull the blade over the sample. Again, repeating this process until a continuous surface is created on the top of the sample.

Now using a paint brush, cover the resulting surface with a corn starch solution. Then lift the sample by 15 microns and place the brush on the surface of the sample. Pull the blades slowly toward the sample beginning a thin cut below the brush and using the brush to guide the section on the surface of the blade when the entire section has been cut.

Use another brush and water to remove the tissue from the blade and place it on a glass slide to allow distinction between the lignified and non lignified structures. Wash away the glycerol with water using a pipette. Next, cover the wet section with a few drops of a mixture of saffron in and astro blue.

After five minutes, wash away the dyes with water and then immediately dehydrate the section with successive ethanol treatments. After the dehydrated ethanol treatment, rinse the section repeatedly in 100%xol. As soon as the xol stays clear.

Cover the tissue with a drop of 100%Canada balsam and a cover glass taking care to avoid air bubbles. Then place the resulting micro slide between two strips of heat resistant plastic and place it on a metal plate. Place a weight on top of the slide to keep the section flat, and then place the slide in a 60 degree Celsius oven.

After 12 hours, allow the slide to cool to room temperature, and then remove the weight and plastic strip. Finally, clean the slide using razor blades to remove any surplus balsam by following the sectioning and staining procedures presented before one can prepare all kinds of samples of different sizes and forms, also by using special holders for course to create digital images of the core surfaces for vessel analysis. First, cut the core surface plane with the core microtome as just demonstrated, and use a felt marker to stain the resulting surface black.

As soon as the dye dries, rub the core surface with white chalk and then press the chalk into the cells with a finger. Remove any surplus chalk, and then place the core on the stage of a binocular microscope equipped with a digital camera starting on one side of the core. Now acquire a sequence of slightly overlapping images until the entire surface is captured.

Then stitch the single images together to create a complete image of the core surface to create digital images from the micro slides. Place the slide on the microscope stage and choose the appropriate magnification for the structures to be analyzed. Finally, acquire overlapping images of the sections mounted on the cleaned micro slides as just demonstrated for the core surface sample and stitch the images together to create one complete image of the section.

The ability to have open cells is a first important step towards the adaption of anatomical dendro ecological structures into a time series analysis. For a more detailed analysis of early wood or late wood traches in conifers, high quality micro sections are needed here. Potential artifacts such as broken cell walls need to be avoided.

The application of a non-Newtonian fluid to the sample, like the cornstarch solution as just demonstrated supports the stability of the cell structure. Micro sections enable a more secure determination of annual ranks. This is especially true for conifers growing on their natural limits.

As seen here, extremely narrow rings are frequent but hard to detect macroscopically. In extreme cases, the rings consist of one or two cell rows, which may only be visible when using micro sections. When analyzing images at high magnifications, single cells are visible.

Semi-automatic analysis software allows the changes in single parameters such as cell wall thickness to be determined for all of the rings visible within the image, indicating the need for an extended time series analysis. Double staining of the micro sections allows the visualization of the different stages of lignification. This information can then be related to the environmental data of the respective vegetation period to determine a more detailed climate growth relationship After its development.

This technique paved the way for researchers in the field of DRO ecology to explore the various anatomical parameters of trees and shrubs. Using this method drastically reduces the time needed to produce big, high quality sections. Precautions such as working below a fume hood and wearing gloves should always be taken while performing this procedure.

After watching this video, you should have a good understanding of how to generate well replicated and highly resolved to put anatomical data sets to support time series analysis.