preparing the mounted cores in the lab for digitizing the cores

<meta charset="utf8"></head><body>Protocolo para digitalização de anéis de árvores de campo para laboratório em estudos dendroecológicos

The principle of dating tree rings by applying the cross-dating technique was introduced the first time by the Austrian forest scientist Arthur Freiherr von Seckendorff-Gudent in 18811. In the first half of the 20th century, this technique was reinvented by the &#34;Father of Dendrochronology&#34; Andrew Ellicott Douglass, who intensively applied it in dating archaeological sites and living trees2.
Nowadays, dendroecology, the research topic acting as a kind of environmental frame of dendrochronology, is defined as the study of tree rings and their inherent growth variations caused by ecological and environmental changes in time3. In dendroecological research, many other characteristics than ring-width variations, such as stable isotopes, late wood density, or cell characteristics within single rings, are used to correlate these data to environmental parameters to better understand the impact of environmental conditions on tree growth over time4. Through the ongoing integration of wood anatomical studies to dendroecological research, dendroecology research evolved in the last decade and is more than ever a backbone in reconstructing past climate conditions5,6,7,8.
Although the technical development regarding sample preparation and analysis, especially in wood anatomy, was strong in the last decade9,10,11,12,13,14, there was almost no real advance regarding the simplification of sampling techniques15. Despite, e.g., acoustic wave technology16, until nowadays there is no reliable &#34;non-destructive&#34; method to extract the characteristics of rings from trees.
Consequently, all tree-ring-related studies still rely on wooden samples taken from trees or shrubs taken at the sites of interest. When focusing on trees, the standard procedure is taking increment cores from stems15.
Taking cores by using increment corers is frequently expressed as a &#34;non-destructive&#34; technique17. Compared to taking disks from stems, this is correct; nevertheless, this sampling technique causes a hole in the stem of about 1 cm in diameter, mostly reaching beyond the pith of the stem3. The tree is able to close this wound on its own, but this process causes growth reactions, altering the common structure in the close vicinity of the wound as well as a more or less intense discoloration of the existing wood around the hole because of fungal diseases18,19. So, it should better be called &#34;minimally invasive&#34; rather than &#34;non-destructive&#34;.
The technique of taking increment cores evolved recently through the ability to use mechanical drills, resulting in higher quality samples, especially for wood anatomical analyses15. This procedure also saves a lot of time in the field compared to manual coring. What remained unchanged was the procedure of handling the cores, starting from the extraction from the tree to labeling, storing for transport, and preparing them in the lab for various possible analyzing techniques.
Cores still need to be packed in stable containers, such as straws made of plastics or paper, to prevent them from breakage during transport. Labeling the cores is done directly on the core using soft pencils or (more frequently) on the outside of each straw. When using plastic containers, the cores must be taken out after a short time to avoid the spread of fungi. So, the cores need to be taken out of the containers again. To stabilize the cores and to prevent them from bending when they start drying, the cores need to be fixed on a mount. This also helps with the subsequent surface preparation for further analyses. When doing so, the labels also need to be transferred to the respective mounts. A standard procedure is gluing the cores on wooden mounts or fixing them with tape in the rills of corrugated boards. Gluing them on wooden mounts is the most frequently used technique. Although this procedure is perfect for stabilizing and sanding or cutting the cores, it has several disadvantages regarding potential chemical, isotopic, and even wood anatomical analyses. Another disadvantage, despite the time required, is the error-prone transfer of the labels for each core to the new mounts.
In dendrochronology, ring-width measurements as a base for accurate dating are the backbone of all dendroecological studies20. Although many labs still rely on manual measurements using measurement tables, e.g., Lintab21 with attached binoculars, there is a trend of using flatbed scanners to digitize core surfaces and measure ring-width using software such as CooRecorder22 or WinDENDRO23. Unfortunately, these scanners, e.g., the widely used Epson Expression 10000XL do not have sufficient resolution to clearly depict structures as earlywood or latewood tracheids (Figure 1). For this reason, the resulting images are not suitable for recognizing difficult structures such as very narrow rings or density fluctuations, which are critical for an accurate cross-dating procedure without going back to the original cores using binoculars24,25.
Since high image resolution is an indispensable prerequisite for adequate image analyses in tree-ring science10, a new image-capturing system was developed at WSL (Skippy; https://www.wsl.ch/en/services-produkte/skippy/) to digitize tree rings on core surfaces using a digital camera resulting in images presenting a higher resolution than all existing flatbed scanners. This system was based on the idea of the ATRICS-system26, developed in 2007. Most recently, a simple but efficient image-capturing system comparable to the Skippy was presented as a self-assembly kit27.
Digitizing tree rings, i.e., reflected light image capturing, is an important step in creating high-resolution images of increment cores or disks to support a time-efficient, digitally based ring-width measurement. The system developed at WSL also allows taking images from long micro sections (up to 40 cm) using transmitted light. This additional feature is, for example, of interest for dendrogeomorphic applications to identify the onset of reaction wood in micro sections.
In the study, we present a protocol to ease the process of handling cores in the field and the lab. The base of the new technique presented is a reusable mount; the new GSC-holder G&#228;rtnerSchneiderCore (GSC) holder designed using 3D-modeling software and printed with a 3D printer. The GSC-holder allows for straightforward handling of the cores taken in the field without repacking or relabeling them. We also present an efficient new system for digitizing the prepared surfaces of the cores. This protocol spans the entire procedure from taking cores in the field to sample preparation, digitizing the core surfaces for subsequent analyses, and eventually storing them in an archive.

<meta charset="utf8"></head><body>Autor em destaque: Desenvolvimento de dispositivos inovadores para o avanço da pesquisa em dendroecologia e anatomia da madeira

Um novo fluxo de trabalho para amostragem e digitalização de núcleos de incremento 

By developing new devices we intend to push boundaries in tree ring research. The application of 3D modeling software and 3D printers allows a fast and cheap development of new devices. One of those is the image capturing system, Skippy, which allows digitizing the surface of increment course in high resolution and short time.Usually when sampling in dendroecology, dendrogeomorphology, or dendroclimatology, we sample trees in remote regions like very high latitude regions and where also conditions, climatic conditions are harsh. So saving time is very important and adopting this protocol we will save time, which is money. Applying our protocol will allow to produce bigger data sets in shorter time.

Preparando os núcleos montados no laboratório para digitalizar os núcleos

preparing-the-mounted-cores-in-the-lab-for-digitizing-the-cores

Research

JoVE Journal

Environment

Preparing the Mounted Cores in the Lab for Digitizing the Cores

A New Workflow for Sampling and Digitizing Increment Cores 

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for further analyses for subsequent dendroecological analyses. The procedure involves the use of new sample carriers for increment cores. These new G&#228;rtner Schneider Core (GSC) holders are designed using three-dimensional (3D) modeling software and finally printed with a 3D printer. Using these mounts from the beginning in the field, the cores can directly be cut with a core microtome, and their surface can then be digitized without further rearrangement using a new high-resolution image-capturing system. They are thus available for direct analysis. This system allows digitizing tree rings from cores and disks, and also taking images from long micro sections (up to 40 cm) using transmitted light. This feature is of special interest for dendroecological and geomorphic applications to identify the onset of any disturbance in micro sections cut with a core-microtome.

We present a protocol to use 3D-printed mounts to fix increment cores in the field without the necessity of unpacking and gluing them on wooden mounts. The new GSC holder allows placing the cores in a core microtome to cut their surface and directly transfer them to digital image capturing.

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for ...

Ambiente

Creating the GSC-Holder Using 3D Modeling Software

To begin, open the 3D model of the holder in a slicer program compatible with a 3D printer. Create a print file that can be read by the 3D printer, ensuring it is saved as a G-code file. Using a memory card or a USB stick, activate the print file on the 3D printer.Once the holder is printed, wait until it cools to room temperature. Remove the plate the holder sticks to from the printer. Bend the plate slightly until the holder separates from the surface.Lastly, remove all excess threads or attachments from the holder.

Criando o GSC-Holder usando software de modelagem 3D

Extracting, Stabilizing, and Transporting Increment Cores in the Field

To begin, take a cordless drill with a torque booster and an increment core. After selecting the coring position, place the corer perpendicular to the growing axis of the stem. Then start coring until the core reaches at least half of the stem diameter.Check the depth by holding the extractor alongside the core. If using a cordless drill, remove the drill. Place the handle on the core.Take the extractor with the open side facing upwards and insert it fully into the core. Then turn the increment core backward one full turn to break the core off the stem. After that, take out the extractor, including the core.Now remove the core from the extractor. Check the fiber direction of the core to ensure an upright orientation when placing the core in the holder. Afterward, place the core on top of the holder with the fiber direction upright.Press on top of the core with all fingers until it slides into the holder. Using a soft pencil, label the core on the side of the holder. Place the holder with the core into the transport box, and close the cover of the transport box.

Extraindo, estabilizando e transportando núcleos de incremento no campo

To begin, place a steel box with a lid fitted with a valve for a vacuum pump connection on a hot plate. Fill the box up to approximately two centimeters with paraffin and wait until it has completely melted. Take the mounted cores out of the transport box.Place the holders with the cores in the liquid paraffin and close the lid. Then, start the vacuum pump and apply a constant light vacuum to the container. After stopping the vacuum pump, open the lid.Take out the holders with the cores, place them on a grid and let them cool down. If needed, remove surplus paraffin from the sides of the holder. Remove the mounted cores from the transport box or the paraffin bath.Place the holder with the core in the sample holder of a core microtome. Ensure the latewood of the rings faces toward the blade. Now, tighten the screws of the sample holder until the core holder is completely secure.Lift the sample holder until the core slightly touches the blade. Pull the blade over the entire extent of the core to cut off the first part of the top. Then, push back the knife behind the core, and lift the sample holder a few microns.Once the surface is cut as intended, remove the core holder from the sample holder of the microtome.

Digitizing the Core Surfaces for Analysis and Storage of Cores

To begin, place the core holder with the plain core surface on the table of an image capturing system, such as the WSL Skippy system. Align the core holder with the moving direction of the table or camera. Position the table with the core holder below the camera, so the outermost ring is in the center of view under the camera objective.Now place a scale next to the onset of the core and take an image for calibration purposes. Define the length of the core in the software, and start the image capturing process. Once the last image is taken, the table moves back to the starting position.Then remove the sample from the table. Place the next holder below the camera. Next, use distortion free stitching software, such as PD GUI to combine the single images into one final image of the core surface.Take the analyzed cores in the holder and place them in the portable storage rack printed with a 3D printer. Label the rack to identify the cores from the outside. Store the rack on a shelf, or any other available archive.The high resolution camera system achieved a real resolution of 6, 500 DPI compared to 1, 825 DPI from a flatbed scanner. The camera system provided clear images of single cells enabling precise identification of ring boundaries. The system facilitated imaging of micro sections of tree cores up to 40 centimeters with transmitted light, which is useful for dendrogeomorphology studies.