1. Manual coring
<ol>
	<li>Assemble the increment corer and select the coring position on the stem of a tree depending on the research question (e.g., for geomorphic reconstructions, parallel to the direction of mechanical stress; for age determination, as low down as possible). 
	NOTE: Always take two cores from each stem, preferably in the opposite direction.</li>
	<li>After selecting the coring position, place the corer at a right angle relative to the direction of growth of the stem.</li>
	<li>Place a pusher at the rear end of the corer to stabilize it while drilling.</li>
	<li>Achieve a stable position and lean against the pusher to apply pressure to the cutting edge.</li>
	<li>Turn the handle of the corer with both hands until the threaded part of the drill is fully turned into the stem.</li>
	<li>Release the pressure and remove the pusher.</li>
	<li>Start turning the handle of the corer with both hands until the corer has reached or drilled through the pith. Check this by holding the extractor (which has the same length as the corer) on the handle aside the stem.</li>
	<li>Take the extractor with the open side on top and insert it fully into the corer. Turn the corer backward (one full turn) to break the core from the stem. Pull the extractor out of the corer.</li>
	<li>Remove the core from the extractor and store it in a paper straw.</li>
	<li>Remove the corer from the stem and store it in the handle.</li>
</ol>
2. Coring with a cordless drill
<ol>
	<li>Take the cordless drill equipped with a torque booster and add the special adapter for the increment corer developed at WSL.</li>
	<li>Place the increment corer into the adapter on the torque booster and select the position for coring on the stem of a tree depending on the research task (see step 1.1).</li>
	<li>After selecting the coring position, place the corer at a right angle relative to the direction of growth of the stem.</li>
	<li>Achieve a stable position, hold the cordless drill tight, and apply pressure on the cutting edge.</li>
	<li>Start the cordless drill, turning slowly until the threaded part of the drill is fully cored into the stem, then increase the speed until the corer reaches or drills through the pith. 
	NOTE: The depth can be checked as explained in step 1.7.</li>
	<li>Remove the cordless drill from the corer, place the handle on it, and use the extractor to remove the core as explained in step 1.8.</li>
	<li>Store the increment core in a paper straw.</li>
	<li>Remove the handle, place the cordless drill on the corer and remove the corer from the stem.</li>
</ol>
3. Sharpening the cutting edge of increment corers
<ol>
	<li>Using the WSL sharpening support
	<ol>
		<li>Take the newly designed holder and place it on the ground.</li>
		<li>Place the cordless drill, including the increment corer, at the designated support points and close the mounting bracket to fix the cordless drill.</li>
		<li>Start the cordless drill by fixing the Teflon block on the starter button and let it run. Take the conical grinding stone and grind the inside of the cutting edge with it. 
		NOTE: The contact angle depends on the inside of the cutting edge. The grinding stone should have full contact with the inner sidewall, reaching from the cutting edge until the inner widening of the corer.</li>
		<li>Take the rectangular grindstone and grind the outside of the cutting edge to deburr it. 
		NOTE: This is necessary to remove the previously formed burr on the edge by grinding it from the inside and to finally sharpen the edge.</li>
		<li>Remove the Teflon block from the starter button to stop the drill, open the mounting bracket to release the cordless drill, and take the device out of the holder.</li>
	</ol>
	</li>
	<li>Checking the sharpness of the cutting edge
	<ol>
		<li>Remove the increment corer from the adapter of the cordless drill.</li>
		<li>Place a sheet of paper on the wooden board of the sharpening support.</li>
		<li>Place the cutting edge of the corer on the paper while holding the corer vertically.</li>
		<li>Turn the corer while holding it vertically without applying pressure to the corer-only the weight of the corer should be pressing on the paper.</li>
		<li>Lift the corer and check whether a round piece of the paper has remained inside the cutting edge of the corer. If so, the corer is sharp. If not, repeat the sharpening procedure (step 3.1). Repeat the entire procedure (steps 3.1 and 3.2) if the outside of a core is not smooth.</li>
	</ol>
	</li>
</ol>
5. Cutting micro sections of entire increment cores using a water-soluble tape
<ol>
	<li>Place a long glass slide next to the microtome and add some water in the middle of the slide along its entire length.</li>
	<li>Place the core in the sample holder of a core microtome. 
	NOTE: To cut the section as a true cross section, make sure that the direction of the fibers is upright.</li>
	<li>Lift the sample holder until the core almost touches the edge of the blade. Pull the blade over the core to cut off the top.</li>
	<li>Place the knife at the beginning of the core again, lift the sample about 10 &#181;m, and repeat the cutting procedure until a plane surface of at least 2 mm width is obtained.</li>
	<li>Add a corn starch solution on the cut surface using a brush21.</li>
	<li>Use a piece of cloth to remove the surplus solution from the top of the core.</li>
	<li>Cut a strip of water-soluble tape to the same length as the core; place one side of the tape to the start of the core, with an overlap of approximately 1 cm, with the beginning of the core facing the blade of the microtome.</li>
	<li>Attach the tape to the surface of the core by stroking the tape on the surface using a finger.</li>
	<li>Lift the sample in the microtome by 15-20 &#181;m, lift the overlapping piece of the tape a little, and place the microtome blade at the edge of the core.</li>
	<li>Cut the section while holding the end of the tape.</li>
	<li>Take the tape with the thin section stuck on it and place it with the cut facing down on the line of water of the glass slide prepared in step 5.1.</li>
	<li>After approximately 10 s, start removing the tape using tweezers, by holding the tape on one side and lifting it up, while taking care that the section remains on the glass slide.</li>
	<li>To produce a permanent slide of this section, follow the standard procedures22.</li>
</ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tree Rings and Environment: Dendroecology. , (1996).</li><li>Toole, E. R., Gammage, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+from+increment+borings+in+bottomland+hardwoods.">Damage from increment borings in bottomland hardwoods.</a> Journal of Forestry. 57 (12), 909-911 (1959).</li><li>Grissino-Mayer, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+manual+and+tutorial+for+the+proper+use+of+an+increment+borer.">A manual and tutorial for the proper use of an increment borer.</a> Tree-Ring Research. 59 (2), 63-79 (2003).</li><li>Wunder, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+increment+coring+enhance+tree+decay?+New+insights+from+tomography+assessments.">Does increment coring enhance tree decay? New insights from tomography assessments.</a> Canadian Journal of Forest Research. 43 (8), 711-718 (2013).</li><li>Helcoski, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+significant+increase+in+tree+mortality+following+coring+in+a+temperate+hardwood+forest.">No significant increase in tree mortality following coring in a temperate hardwood forest.</a> Tree-Ring Research. 75 (1), 67-72 (2019).</li><li>Hall, A. A., Bloomberg, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+power-driven+increment+borer.">A power-driven increment borer.</a> The Forestry Chronicle. 60 (6), 356-357 (1984).</li><li>Scott, J. H., Arno, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+power+increment+borer+to+determine+the+age+structure+of+old-growth+conifer+stands.">Using a power increment borer to determine the age structure of old-growth conifer stands.</a> Western Journal of Applied Forestry. 7 (4), 100-102 (1992).</li><li>Krottenthaler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+power-driven+increment+borer+for+sampling+high-density+tropical+wood.">A power-driven increment borer for sampling high-density tropical wood.</a> Dendrochronologia. 36, 40-44 (2015).</li><li>Caetano-Andrade, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+increment+coring+system+for+large+tropical+trees+with+high+wood+densities.">Advances in increment coring system for large tropical trees with high wood densities.</a> Dendrochronologia. 68, 125860 (2021).</li><li>Edwards, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-annual+climate+anomalies+in+northwestern+North+America+following+the+1783-1784+CE+Laki+eruption.">Intra-annual climate anomalies in northwestern North America following the 1783-1784 CE Laki eruption.</a> Journal of Geophysical Research: Atmospheres. 126 (3), 033544 (2021).</li><li>Zhirnova, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+495-year+wood+anatomical+record+of+Siberian+stone+pine+(Pinus+sibirica+Du+Tour)+as+climatic+proxy+on+the+timberline.">A 495-year wood anatomical record of Siberian stone pine (Pinus sibirica Du Tour) as climatic proxy on the timberline.</a> Forests. 13 (2), 247 (2022).</li><li>Maeglin, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Increment Cores: How to Collect, Handle, and Use Them. 25, (1979).</li><li>G&#228;rtner, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technical+perspective+in+modern+tree-ring+research+-+how+to+overcome+dendroecological+and+wood+anatomical+challenges.">A technical perspective in modern tree-ring research - how to overcome dendroecological and wood anatomical challenges.</a> Journal of Visualized Experiments. (95), e52337 (2015).</li><li>G&#228;rtner, H., Banzer, L., Schneider, L., Schweingruber, F. H., Bast, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+micro+sections+of+entire+(dry)+conifer+increment+cores+for+wood+anatomical+time-series+analyses.">Preparing micro sections of entire (dry) conifer increment cores for wood anatomical time-series analyses.</a> Dendrochronologia. 34, 19-23 (2015).</li><li>Rodriguez, D. R. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+wood+anatomy,+density+and+chemistry+profiles+to+understand+the+tree-ring+formation+in+Amazonian+tree+species.">Exploring wood anatomy, density and chemistry profiles to understand the tree-ring formation in Amazonian tree species.</a> Dendrochronologia. 71, 125915 (2022).</li><li>G&#228;rtner, H., Farahat, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cambial+activity+of+Moringaperegrina+(Forssk.)+Fiori+in+arid+environments.">Cambial activity of Moringaperegrina (Forssk.) Fiori in arid environments.</a> Frontiers in Plant Science. 12, 760002 (2021).</li><li>von Arx, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q-NET-a+new+scholarly+network+on+quantitative+wood+anatomy.">Q-NET-a new scholarly network on quantitative wood anatomy.</a> Dendrochronologia. 70, 125890 (2021).</li><li>Seftigen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+dendroanatomy+in+paleoclimatology-a+case+study+on+Picea+engelmannii+from+the+Canadian+Rockies.">Prospects for dendroanatomy in paleoclimatology-a case study on Picea engelmannii from the Canadian Rockies.</a> Climate of the Past. 18 (5), 1151-1168 (2022).</li><li>Matulewski, P., Buchwal, A., G&#228;rtner, H., Jagodzi&#324;ski, A. M., &#268;ufar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+growth+with+blue+rings:+comparison+of+radial+growth+and+wood+anatomy+between+trampled+and+non-trampled+Scots+pine+roots.">Altered growth with blue rings: comparison of radial growth and wood anatomy between trampled and non-trampled Scots pine roots.</a> Dendrochronologia. 72, 125922 (2022).</li><li>Prislan, P., del Castillo, E. M., Skoberne, G., &#352;penko, N., Gri&#269;ar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+protocol+for+wood+and+phloem+formation+analyses.">Sample preparation protocol for wood and phloem formation analyses.</a> Dendrochronologia. 73, 125959 (2022).</li><li>Heinrichs, J. 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The authors declare that they have no competing financial interests.

The significant inclusion of wood anatomy in dendroecological studies23,24,&#160;as well as an intensified exchange among scientists specialized in tree-ring research and wood anatomists25, has opened a wide field of new and in-depth analyses of past environmental conditions. These new studies have opened up new possibilities and questions, but have also given rise to new problems.
The fast development of this new era of &#34;dendroanatomy&#34; requires a high number of samples, which is definitely supported by the use of a cordless drill as explained before. In addition to the fact that taking cores with the drill is not at all exhausting, it saves a lot of time. Although the results presented in this paper imply sampling possibilities that are six times faster than manual coring, it is a test for single cores. Nevertheless, during regular sampling (one person coring, with one coding and storing the cores), we managed to core 24 spruce trees (two cores of full length each), with stem diameters of approximately 80 cm, within 1.5 h. This is an average of &#60;2 min for one core, including storage, packing, and moving to the next tree.
The fast handling of the entire process is supported by the fact that the newly designed adapter for increment corers can be used without the need to fix the corer inside the adapter with a screw or comparable closures. As a result, changing the drill to the handle of the corer to break and extract the core is fast and easy. The adapter is designed so that one can even pull the corer out while drilling back in case the stem is rotten, or (as is common with some increment corers) if the thread does not grab when turning back and the corer does not move out.
However, it should be noted that when removing the corer from the stem, one needs to tilt the adapter slightly so that it can be successfully pulled without the drill slipping off (protocol step 2.8). The increasing demand for tree-ring studies to create long chronologies based on anatomical proxies19,26 has required the preparation of micro sections from increment cores, cut in pieces before preparation or cut as entire micro sections22. Although the quality of up to 40 cm long micro sections is still not always comparable to short sections (e.g., the variable angle of the cells in their vertical extension frequently hampers cell wall measurements), they can be used to identify and date specific growth reactions as the occurrence of reaction wood or blue rings27 (Figure 6).
Consequently, the quality of the samples is a basic prerequisite for successful preparation and further analyses of anatomical structures. This demand requires more caution regarding the sharpness of the sampling campaign when taking increment cores. As a consequence, preparing micro sections can be very time-consuming and labor-intensive, and sometimes even impossible, if the specimens are not embedded beforehand28.
Sharpening the cutting edge of an increment corer by hand requires a lot of practice and experience, in order to grind the edge evenly all around by hand without any support. The ability of using the new drill mount for sharpening increment cores enables even users inexperienced in sharpening to sharpen the cutting edge of their corers in the field. The fact that this now can be done quickly will increase the quality of samples taken in future.
Although the use of the new equipment shows clear advantages for subsequent processing of the cores, the cordless drill could also be combined with small devices for sharpening, developed and presented almost 40 years ago20. Maeglin20 presented construction details of a modification of &#34;Goodchild&#39;s borer sharpener&#34; made of wood and metal29. Nowadays, this device can be modeled and printed in a 3D printer without any problems30. One would only need to create a detailed 3D model of the sharpener to print the single parts and assemble it to use in the field. The possibilities for improvement have not yet been exhausted and we are sure that this publication will inspire many colleagues to further develop the tools presented here. A yet unsolved obstacle is the fact that one needs to remove the drill and add the handle of the corer to extract the core.
The final step of cutting micro sections of entire increment cores22 is still a tricky issue. The application of the water-soluble tape, as described earlier, supports the process by stabilizing the section when cutting and placing it on the glass slide. Nevertheless, this procedure still requires of the user to have a high level of experience.

Dendroecological research is based on various characteristics of growth rings in trees, both annual and otherwise. &#34;Precursory&#34; discipline dendrochronology was established using ring-width variations as a parameter to simply date the rings and, as a result, establish long chronologies. Therefore, manifold other characteristics, such as density variations, isotopic concentrations, or wood anatomical characteristics, are used to correlate single rings or their structure and content to environmental parameters to better understand the impact of environmental conditions on tree growth over time.
Dendroecology, as well as dendroclimatology, has gained in importance in environmental research, mainly in reconstructing past climate conditions1,2,3. For this, the rings of countless trees have to be analyzed in detail. Although some techniques exist to determine tree-ring width and density (e.g., by acoustic wave technology4 or drilling resistance5,6), to date, there is no reliable &#34;non-destructive&#34; method to extract the characteristics of rings from trees. For very detailed analyses of ring characteristics within a tree, or to estimate basal area increment, it would be best to cut disks from the trees of interest7. This would require cutting down all potential trees of interest for specific analyses. Bearing in mind the huge number of trees analyzed worldwide each year, this sampling strategy is not practicable. Regardless of wasting an incredible amount of resources, this strategy is simply too expensive. Due to this, the use of increment corers has been established as a standard sampling technique in tree-ring research8. The use of increment corers allows for a minimally invasive extraction of wood cores from stems, starting from the bark and reaching (in optimal cases) the pith of the tree9.
Although coring causes an injury to the stem-a hole with a diameter of ~1 cm-trees are able to close this wound through increased wood formation in the vicinity of the core hole. A disadvantage, apart from the hole itself, is the occurrence of a &#34;compartmentalization zone&#34;, an area around the hole where the cells are filled by phenols to prevent the potential spread of fungi starting from the hole10,11. To our knowledge, there is still no evidence that increment coring causes a significant increase of tree decay frequency, at least in undisturbed high-elevation forest stands for Picea abies12 and several hardwood species in a temperate forest13.
Although this sampling standard has been applied for decades all over the world, some problems still remain. One of these is the fact that the cores have to be taken by hand without any mechanical support, which takes a lot of time and is quite exhausting after a while. To ease sampling, several (more or less practicable) strategies have been tested, such as the use of chainsaws equipped with a corer instead of the chain14,15,16,17. The use of chainsaws was preferred to drills because the latter were not powerful enough; however, this idea did not catch on due to the large weight of the chainsaw and the fuel required.
In recent years, wood anatomical techniques have evolved significantly and been integrated into dendroecological studies18,19. However, the ability to analyze wood anatomical parameters over long periods by cutting micro sections from increment cores resulted in unexpected problems. Frequently, the micro sections taken from cores broke into little pieces, which made it impossible to produce coherent cuts (Figure 1). This problem was caused by the manual technique of coring trees and unsharp corers. The mechanical stress exerted on the wood while coring resulted in micro cracks within the core. These micro cracks were never noticed during macroscopic examination of the increment cores, and therefore never presented a problem.
Manual coring is done by placing the handle on the rear end of the corer, pressing the tip with the thread to the stem, and starting to turn the handle until the corer has pierced a little over half the diameter of the stem. While doing this, the tip of the corer is (obviously) fixed in the stem, but the rear end of the corer turned by the handle is always moving sideward or up and down, at least until the drill head is fully screwed into the trunk, giving more guidance and stability to the corer. As a result of the high pressure and the movement of the corer, the increment cores are distorted frequently in the outermost ~5 cm (Figure 1). Even if the friction while turning is reduced to a minimum, another process is exerting stress on the increment core inside the corer. Manual coring does not allow a continuous movement of the cutting edge of the corer inside the stem. One can do a maximum of one full turn, before having to stop to change the grip, and then continue drilling. Each time the rotation restarts, the core is slightly twisted until the friction is overcome and the drill rotates again. These mechanical stresses potentially cause microscopic cracks in the structure of the cores.
This mechanical stress is even increased when the cutting edge of the corer is not sharp. A visible sign for an unsharp corer is an uneven core surface, showing lots of cracks along its entire extension20 (Figure 2). The frequency of sharpening depends on the density of the trees to be cored and the minerals or sand present in the bark of the tree to be cored. On a general note, one should not assume that new corers are sharp. To date, sharpening a corer is almost never done in the field due to the difficulty of it, since this has to be done by hand and needs a lot of experience11,20.
To summarize, manual coring and unsharp cutting edges both result in micro cracks occurring in cores taken. To date, these problems have not been analyzed systematically, nor have attempts been made to find solutions. This paper presents a protocol to overcome these obstacles by comparing the manual coring technique to the application of a new technique. We propose using a cordless drill equipped with a special adapter for an increment corer. We present to which extent problems are minimized when coring a tree, as well as the effect of continuous, mechanical coring on the preparation of long micro sections. This protocol includes the preparation of long micro sections using a water-soluble tape as a supporting aid and a procedure to sharpen corers in the field.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BS 18 LTX-3 BL QI</td>
			<td>Metabo</td>
			<td>0</td>
			<td>Cordless drill</td>
		</tr>
		<tr>
			<td>Core-microtome</td>
			<td>WSL</td>
			<td>0</td>
			<td>Microtome to cut micro sections from increment cores</td>
		</tr>
		<tr>
			<td>Drill adapter for increment corer</td>
			<td>WSL</td>
			<td>0</td>
			<td>Adapter to fix the increment corer on the cordless drill</td>
		</tr>
		<tr>
			<td>Increment corer</td>
			<td>Hagl&#246;ff</td>
			<td>0</td>
			<td>40cm increment corer</td>
		</tr>
		<tr>
			<td>Power X3</td>
			<td>Metabo</td>
			<td>0</td>
			<td>Torque amplifyer</td>
		</tr>
		<tr>
			<td>Sharpening support board</td>
			<td>WSL</td>
			<td>0</td>
			<td>Board to attach the cordless dril to sharpen the cutting edge ofd the corer</td>
		</tr>
		<tr>
			<td>Water-soluble tape 5414, transparent 3/4IN</td>
			<td>3M</td>
			<td>0</td>
			<td>Transparent tape to support cutting long sections</td>
		</tr>
	</tbody>
</table>

advanced workflow for taking high-quality increment cores - new techniques and devices

In dendroecological research, precise dating of each single growth ring is a basic requirement for all studies, focusing on ring-width variations only, chemical or isotope analyses, or wood anatomical studies. Independent of the sampling strategy for a certain study (e.g., climatology, geomorphology), the way samples are taken is crucial for their successful preparation and analyses.
Until recently, it was sufficient to use a (more or less) sharp increment corer to obtain core samples that could be sanded for further analyses. Since wood anatomical characteristics can be applied to long time series, the need to obtain high-quality increment cores has taken on a new meaning. Essentially, the corer needs to be sharp(ened) when used. When coring a tree by hand, there are some problems in handling the corer, resulting in the hidden occurrence of micro cracks along the entire core: When starting to drill by hand, the drill bit is strongly pressed against the bark and the outermost ring until the thread has fully entered the trunk. At the same time, the drill bit is moved up and down as well as sideward. Then, the corer is drilled all the way into the trunk; however, it is necessary to stop after each turn, change the grip, and turn again. All these movements, as well as the start/stop-coring, puts mechanical stress on the core. The resulting micro cracks make it impossible to create continuous micro sections, as they fall apart along all these cracks.
We present a protocol to overcome these obstacles by applying a new technique using a cordless drill to minimize these problems when coring a tree, as well as its effect on the preparation of long micro sections. This protocol includes the preparation of long micro sections, as well as a procedure to sharpen corers in the field.

When comparing the manual coring procedure to the use of a cordless drill, the advantages of the latter are obvious. We compared coring spruce trees (Picea abies (L.) H. Karst.) with a stem diameter at a breast height of 60-80 cm. We used 5 mm corers, with a length of 40 cm, for all cores taken, and drilled the full length of the corer into the stem. When taking cores manually, the full procedure of taking a core and removing the corer from the tree again took an average of ~6 min. When repeating this using the cordless drill equipped with a torque booster, the entire procedure took an average of only 1 min. In addition to the fact that coring with the cordless drill is not at all exhausting, none of the cores were deformed, due to pressure exerted on the cutting edge during the first phase of drilling until the thread is completely inside the stem. As soon as the thread is inside the stem, the corer is more or less stabilized, and potential up- and downward movements are minimized (Figure 3).
As soon as the first core was no longer smooth on the outside but showed scratches and cracks, as in Figure 2, sharpening of the cutting edge was required. Because the cordless drill can be fixed, as it is used for coring (i.e., including the adapter and the increment corer; Figure&#160;4), the sharpening procedure is also quite fast. With a little practice, sharpening takes no more than 5 min. As soon as the cut paper sticks inside the corer, sampling can be continued. The resulting cores are smooth without any scratches or cracks. The cores taken with the aid of the cordless drill have a lower probability of showing micro cracks; this is a precondition to cut micro sections of entire increment cores. The application of the water-soluble tape (Figure 5) made the handling of the long and fragile sections easier, since the tape protects the thin section from tearing while taking it off the blade and placing it on the glass slide. This procedure saves time in the lab and improves the quality of the micro sections, because the tape glue stabilizes the cell walls while cutting in addition to the non-Newtonian fluid (corn starch solution; see protocol step 5.5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64747/64747fig01.jpg" /> 
Figure 1: Increment corer. (A) Increment corer, as used for manual coring, and a magnified view of the thread and the cutting edge. (B) Distorted increment core due to high pressure exerted to the wood at the beginning of manual coring. (C) Micro section of a part of an increment core fragmented due to micro cracks. Scale bar = 0.5 cm. (D-F) Photos indicating the procedure of coring when using a cordless drill. No high pressure is needed to start coring (D,E), the handle can easily be used to extract the core (F), and the drill is extracted directly thereafter (G). <a href="https://www.jove.com/files/ftp_upload/64747/64747fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64747/64747fig02.jpg" /> 
Figure 2: Increment core showing scratches and cracks on the outside due to the use of an unsharp corer. Scale bar = 0.5 cm. <a href="https://www.jove.com/files/ftp_upload/64747/64747fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64747/64747fig03.jpg" /> 
Figure 3: Increment cores and related micro sections. (A) Straight core taken with a sharp corer and a cordless drill. (B) Distorted core taken manually with an unsharp corer. (C) Continuous section of a Pinus sylvestris core sampled with a sharp corer. (D) Section of a Larix decidua core broken in pieces due to using an unsharp corer. Scale bars = 0.5 cm (A,B); 1 cm (C). <a href="https://www.jove.com/files/ftp_upload/64747/64747fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64747/64747fig04.jpg" /> 
Figure 4: Holder designed for sharpening the cutting edge of increment corers. (A)&#160;Teflon block to guide and stabilize the increment corer. (B) Teflon block to fix the starter of the cordless drill. (C) Hexagon wrench to fix A at other positions on the board, depending on the type of corer used. (D) Clamp to fix the cordless drill. (E) Placing the conical grinding stone inside the cutting edge. (F) Placing the straight grinding stone outside to deburr the cutting edge. <a href="https://www.jove.com/files/ftp_upload/64747/64747fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64747/64747fig05.jpg" /> 
Figure 5: Application of the water-soluble tape. (A) Cut the tape to the length needed to cover the core surface. (B) Place the tape on the prepared surface of the core. (C) Cut the section by holding the edge of the tape in one hand. (D) Place the tape with the section facing down on a glass slide and add water to separate the tape from the section. <a href="https://www.jove.com/files/ftp_upload/64747/64747fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64747/64747fig06.jpg" /> 
Figure 6: Micro section of a conifer showing a &#34;blue ring&#34;. Latewood cell walls not lignified and, for this, blue, in the ring of the year 1974 (magnified above the slide showing the long section). Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/64747/64747fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advanced Workflow for Taking High-Quality Increment Cores - New Techniques and Devices at JoVE.com

Advanced Workflow for Taking High-Quality Increment Cores - New Techniques and Devices

Here, we present a protocol on how to avoid micro cracks in increment cores by applying a cordless drill with a torque multiplier to minimize problems when coring trees, as well as its effect on preparing long micro sections. This protocol also includes a procedure to sharpen corers in the field.

In dendroecological research, precise dating of each single growth ring is a basic requirement for all studies, focusing on ring-width variations only, ...

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1.8K Views.  Swiss Federal Research Institute WSL. Here, we present a protocol on how to avoid micro cracks in increment cores by applying a cordless drill with a torque multiplier to minimize problems when coring trees, as well as its effect on preparing long micro sections. This protocol also includes a procedure to sharpen corers in the field.

Video: Advanced Workflow for Taking High-Quality Increment Cores - New Techniques and Devices

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are resistant to low molecular-weight oil components, unlike traditional polydimethylsiloxane (PDMS) devices. Here, we demonstrate a facile method for making a device with this property, and we use the product of this protocol for examining the pore-scale mechanisms by which foam recovers crude oil. A pattern is first designed using computer-aided design (CAD) software and printed on a transparency with a high-resolution printer. This pattern is then transferred to a photoresist via a lithography procedure. PDMS is cast on the pattern, cured in an oven, and removed to obtain a mold. A thiol-ene crosslinking polymer, commonly used as an optical adhesive (OA), is then poured onto the mold and cured under UV light. The PDMS mold is peeled away from the optical adhesive cast. A glass substrate is then prepared, and the two halves of the device are bonded together. Optical adhesive-based devices are more robust than traditional PDMS microfluidic devices. The epoxy structure is resistant to swelling by many organic solvents, which opens new possibilities for experiments involving light organic liquids. Additionally, the surface wettability behavior of these devices is more stable than that of PDMS. The construction of optical adhesive microfluidic devices is simple, yet requires incrementally more effort than the making of PDMS-based devices. Also, though optical adhesive devices are stable in organic liquids, they may exhibit reduced bond-strength after a long time. Optical adhesive microfluidic devices can be made in geometries that act as 2-D micromodels for porous media. These devices are applied in the study of oil displacement to improve our understanding of the pore-scale mechanisms involved in enhanced oil recovery and aquifer remediation.

The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported.

Microfluidic devices are versatile tools for studying transport processes at a microscopic scale. A demand exists for microfluidic devices that are ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has become particularly relevant for assessing the anthropogenic-altered global nitrogen cycle and the emission of greenhouse gases (i.e., N2O). Several methods are available for measuring denitrification, but none of them are completely satisfactory. Problems with existing methods include their insufficient sensitivity, and the need to modify the substrate levels or alter the physical configuration of the process using disturbed samples.This work describes a method for estimating sediment denitrification rates that combines coring, acetylene inhibition, and microsensor measurements of the accumulated N2O. The main advantages of this method are a low disturbance of the sediment structure and the collection of a continuous record of N2O accumulation; these enable estimates of reliable denitrification rates with minimum values up to 0.4-1 &#181;mol N2O m-2 h-1. The ability to manipulate key factors is an additional advantage for obtaining experimental insights. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate. The method is appropriate for estimating denitrification rates in any aquatic system with retrievable sediment cores. If the N2O concentration is above the detection limit of the sensor, the acetylene inhibition step can be omitted to estimate the N2O emission instead of denitrification. We show how to estimate both actual and potential denitrification rates by increasing nitrate availability as well as the temperature dependence of the process. We illustrate the procedure using mountain lake sediments and discuss the advantages and weaknesses of the technique compared to other methods. This method can be modified for particular purposes; for instance, it can be combined with 15N tracers to assess nitrification and denitrification or field in situ measurements of denitrification rates.

Estimating Sediment Denitrification Rates Using Cores and N2O Microsensors

This method estimates sediment denitrification rates in sediment cores using the acetylene inhibition technique and microsensor measurements of the accumulated N2O. The protocol describes procedures for collecting the cores, calibrating the sensors, performing the acetylene inhibition, measuring the N2O accumulation, and calculating the denitrification rate.

Denitrification is the primary biogeochemical process removing reactive nitrogen from the biosphere. The quantitative evaluation of this process has ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The power of their influence is in extraradical mycelia that spread beyond nutrient depletion zones found near roots to ultimately interconnect individuals within a common mycorrhizal network (CMN). Most experiments, however, have investigated the role of AM fungi in plant interactions by growing plants with versus without mycorrhizal fungi, a method that fails to explicitly address the role of CMNs. Here, we propose a method that manipulates CMNs to investigate their role in plant interactions. Our method uses modified containers with conical bottoms with a nylon mesh and/or hydrophobic material covering slotted openings, 15N fertilizer, and a nutrient-poor interstitial sand. CMNs are left either intact between interacting individuals, severed by rotation of containers, or prevented from forming by a solid barrier. Our findings suggest that rotating containers is sufficient to disrupt CMNs and prevent their effects on plant interactions across CMNs. Our approach is advantageous because it mimics aspects of nature, such as seedlings tapping into already established CMNs and the use of a suite of AM fungi that may provide diverse benefits. Although our experiment is limited to investigating plants at the seedling stage, plant interactions across CMNs can be detected using our approach which therefore can be applied to investigate biological questions about the functioning of CMNs in ecosystems.

Most plants within communities likely are interconnected by arbuscular mycorrhizal (AM) fungi, but mediation of plant interactions by them has been investigated primarily by growing plants with versus without mycorrhizas. We present a method to manipulate common mycorrhizal networks among mycorrhizal plants to investigate their consequences for plant interactions.

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

An Application for Pairing with Wearable Devices to Monitor Personal Health Status

The current protocol aims to showcase the technology integration, providing a detailed description of adopting the HealthCloud app, developed by the Healthy Landscape and Healthy People Lab, National Taiwan University (HLHP-NTU), on smartphones and smartwatches to collect data on users' real-time psychological and physiological responses and environmental information. A flexible and integrated research method was proposed because it can be difficult to measure multi-dimensional aspects of personal data in on-site studies in landscape and outdoor recreation research. An on-site study conducted in 2020 at the National Taiwan University campus was used as an application example. A dataset of 385 participants was used after excluding invalid samples. During the experiment, participants were asked to walk around campus for 30 min when their heart rate and psychological-scale items were measured, together with several environmental metrics. This work aimed to provide a possible solution to help on-site studies track real-time human responses that match ambient factors. Due to the app's flexibility, its use on wearable devices shows excellent potential for multidisciplinary research studies.

The present protocol introduces a non-commercial self-developed application for collecting real-time on-site data, including psychological scales, GPS location, heart rate, and blood-oxygen saturation level, as well as the application's operating procedures. An empirical study conducted in Taiwan in 2020 was used as an application example.

The current protocol aims to showcase the technology integration, providing a detailed description of adopting the HealthCloud app, developed by the ...