This work was supported by the Fundamental Research Funds for the Central Universities (2017ZY21) and the National Natural Science Foundation of China (21607038).

1. Leaf Collection, Elution and Mass Measurement of PM
<ol>
	<li>Select five healthy individual trees (i.e., five replicates) of each tree species with similar diameter at breast height. Collect four larger branches randomly from four directions of the outer canopy in the middle canopy layer and cut off all intact leaves. 
	NOTE: All plants for leaf sampling should be located closely in a greening strip with length and width of about 250 and 60 m, respectively, to ensure that the environment conditions (wind, light, and rain) of these trees are similar. The leaves used in the protocol were collected on October 15th (short dust retention (SDR) period) and October 25th (long dust retention (LDR) period) in 2014, which were 4 and 14 days after the latest rainfall (&#62; 15 mm), respectively. The average levels of PM under the short and long dust retention period (i.e., the duration between the last rainfall and the leaf sampling time) in our experiment were 26 (PM2.5), 57 (PM10), and 111 (PM2.5), 160 &#181;g/m3 (PM10), respectively.
	<ol>
		<li>Place the sampled leaves in labeled valve bags and transport the bags to the laboratory immediately. Store the leaf samples in the fridge.</li>
	</ol>
	</li>
	<li>Wash and dry the beakers in the 80 &#176;C oven. Equilibrate the beakers to room temperature and humidity and weigh the empty beakers (W1).</li>
	<li>Randomly select a certain amount of leaves from leaf samples and put the leaves in a 1000 mL beaker (Beaker A). 
	NOTE: The leaf area is about 2000 cm2, which can guarantee all the leaves can be immersed in the water completely and the eluted dust has sufficient weight to be weighed accurately.</li>
	<li>Add 270 mL of deionized water to the Beaker A and immerse the leaves in water completely.
	<ol>
		<li>Stir the water for 60 s with a glass rod in one direction (frequency: 2 seconds for one rotation). Afterwards, pour the eluent into three 100 mL small beakers (Beaker a) evenly.</li>
		<li>Wash the leaves using a fine tipped squeeze bottle with 30 mL of deionized water and transfer the washed leaves to a 1000 mL beaker (Beaker B). Pour the eluent into three 100 mL small beaker (Beaker a) evenly.</li>
	</ol>
	</li>
	<li>Add 270 mL of deionized water to Beaker B and immerse the leaves in water again. Then use a nylon brush to scrub the leaf surface (placing on flat thin plastic plate) with deionized water and avoid destroying the microstructure of leaf surface. Pour the eluent into three 100 mL small beakers (Beaker b).
	<ol>
		<li>Wash the leaves using the squeezable bottle with fine tip with 30 mL of deionized water and transfer the leaves into a 1000 mL beaker (Beaker C). Pour the eluent into three 100 mL small beaker (Beaker b).</li>
	</ol>
	</li>
	<li>Add 270 mL of deionized water to Beaker C and immerse the leaves in water again.
	<ol>
		<li>Put the glass container into the ultrasonic cleaning machine. Using an ultrasonic power of 500 W, clean for 3 min and 10 min for leaves of the broadleaf and needleleaf tree species, respectively. Stir the leaves with a glass rod in one direction (frequency: 2 seconds for one circle) simultaneously.</li>
		<li>Wash the leaves using the squeezable bottle with fine tip with 30 mL of deionized water and pour the eluent into three 100 mL small beakers (beaker c).</li>
	</ol>
	</li>
	<li>Cover a piece of clean filter paper (diameter = 11 cm, area = 94.99 cm2) on each beaker (a, b, c) and dry the beakers in the 80 &#176;C oven for approximately 5 days until the mass of the beakers becomes constant.
	<ol>
		<li>Put the beakers in a balance chamber to equilibrate the temperature and humidity for 30 min, and weigh the mass of each 100 mL beakers (W2). Calculate the mass of PM eluted by each cleaning step by W2-W1.</li>
	</ol>
	</li>
</ol>
2. Measurement of PM Size Distribution and Leaf Area
<ol>
	<li>Add 50 mL of deionized water to each weighed beaker (a, b, c) mentioned above and place these beakers in an ultrasonic cleaning machine for 30 min until the PM disperses in deionized water.</li>
	<li>Add the supernatant in beaker (a, b, c) to the laser granularity instrument and measure the size distribution of PM eluted by different cleaning steps.
	<ol>
		<li>Assume the measured volume percentages to be mass percentages (Q) of different-sized particles. Calculate the proportion of different-sized particles eluted by each cleaning step by equation (1): 
		<img alt="Equation 1" src="/files/ftp_upload/58026/58026eq1.jpg" />&#160; &#160;(1) 
		where Pi,j represents the mass proportion (%) of the particles within the j diameter class eluted from the leaf surfaces by the cleaning step i; Wi represents the total mass (g) of all sized particles eluted by the cleaning step i; Qi,j represents the mass percentage (%) of the particles within the j diameter class in the total PM mass eluted by the cleaning step i; i is the cleaning step (i.e., WC, BC, and UC); and j is the diameter class, which was set to d &#8804; 1 &#181;m (PM1), 1 &#60; d &#8804; 2.5 &#181;m (PM1-2.5), 2.5 &#60; d &#8804; 5 &#181;m (PM2.5-5), 5 &#60; d &#8804; 10 &#181;m (PM5-10), d &#62; 10 &#181;m (PM&#62;10) in the present study.</li>
	</ol>
	</li>
	<li>Spread leaves on the plastic board and scan the leaves with a high-quality scanner. Use automatic image analysis software to estimate the surface area and projected area of leaves. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Data Presentation and Analysis
<ol>
	<li>Calculate the total removable particulate matter (TRP) as the sum of the ERP and DRP that can be eluted by WC + BC + UC.</li>
	<li>Under different dust retention periods, calculate the total mass of the PM within a specificdiameter class retained on leaves as the sum of the mass of the PM within corresponding diameter class eluted by the different cleaning steps (i.e., WC, BC, and UC).
	<ol>
		<li>Using these data and the leaf area data, calculate the retention efficiency (AEleaf) of the various sized particles on unit leaf surface area using equation (2): 
		<img alt="Equation 2" src="/files/ftp_upload/58026/58026eq2.jpg" />&#160; &#160; &#160;(2) 
		where LZj and SZj&#160;are the mass (g) of the particles within the j diameter class retained on unit leaf area under the periods of LDR and SDR, respectively; LT and ST are the numbers of days in the periods of LDR and SDR, respectively.</li>
	</ol>
	</li>
	<li>Conduct all the statistical analyses with SPSS software.
	<ol>
		<li>Use the Kolmogorov-Smirnov test and the Levene test to verify the ANOVA assumptions of normality and the homogeneity of variances, respectively, for the elution percentages of the different-sized particles and the PM retention capacity data.</li>
		<li>Apply one-way ANOVA to investigate the effects of the different cleaning steps on the elution percentages of the different-sized particles under various dust retention periods. Use Duncan&#39;s test (P = 0.05) to detect the significant differences among different cleaning steps.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Accurate and proper collection of the PM retained on leaf surfaces is the basis for assessing the PM removal abilities of different tree species. However, the conventional cleaning method (WC or plus BC) cannot completely remove the dust on leaf surfaces, which has been confirmed by scanning electron microscopy10. This was further demonstrated clearly by the present study (Figure 1, Figure 2,&#160;Figure 3, Figure 4, and&#160;Figure 5). Our study shows that, if only WC was applied to clean leaves, the PM on leaf surface would be underestimated by about 69% and 65% under the SDR and LDR periods, respectively. That is to say, a short period of rainfall with a certain intensity could only elute 31% and 35% of PM from leaf surfaces. In addition, previous studies have shown that a short and heavy rainfall could only elute 50% and 62% of the PM from the leaves of Ligustrum lucidum and Viburnum odoratissimum, respectively18. However, influence of rainfall on eluting the PM retained on leaf surfaces of P. tabuliformis was not evident. Consequently, under natural conditions, only a small part of PM on leaf surface could be eluted by rainfall. When both WC and BC were applied to clean leaves, the elution percentage of PM would also be underestimated by about 41% and 36% under the SDR and LDR periods, respectively. However, more PM could be eluted and collected from leaf surface after supplementing UC to WC+BC for cleaning leaves. Thus, for unbiased and accurate quantification of PM retained by leaves, it is necessary and crucial to add UC to the conventional leaf cleaning method.
At present, most studies using the retention capacity of PM retained on leaf surfaces to assess the particulate matter removal abilities of trees. Although this indicator is convenient for assessing the PM removal abilities under the same retention duration, there will be great difference in the retention capacities of the same tree species under different dust retention durations. Thus, it was proposed in some studies that the retention efficiency (the mass of PM retained on a unit of leaf area per unit time) should be applied to assess the plant PM removal abilities, as this can eliminate the evaluation deviation of PM removal ability due to the difference in dust retention duration. However, these studies neglected the fact that only a small part of PM on leaf surface can be eluted by rainfall. In addition, this method could result in five times overestimation of the PM removal abilities of trees, according to the results of this study (Table 1). For this reason, the calculation method of equation (2) should be applied to accurately assess the PM removal abilities of trees.
When PM elution method proposed in this study was used to determine the mass of PM on leaf surfaces, each experimental step must be accurate and avoid the errors caused by human factors as much as possible. For example, the number of leaves for each experiment depends on the specific situation, and it should be determined by the specification of experiment instrument, the dust retention duration, ultrasonic parameter, and other factors. Taking ultrasonic parameter as example, the ultrasonic cleaning duration and the centrifuge speed should be determined by multiple preliminary experiments to ensure the experimental error is within the acceptable range. In addition, the amount of deionized water used for elution should also be adjusted according to the mass of PM. Besides, each small beaker filled with eluent, while it is drying in the oven, should be covered with a piece of clean filter paper to prevent dust pollution. In brief, each step in the experiment must be carefully operated in order to repeat the proposed method accurately.
It is extremely necessary and crucial to supplement the ultrasonic cleaning procedure to the conventional leaf cleaning method, so that the PM removal abilities of trees can be assessed more accurately and quantitatively. In order to accurately compare the PM removal abilities of different tree species experiencing different dust retention durations, the retention efficiency should be calculated using the method (equation (2)) proposed in this study. Our proposed comprehensive protocol will be useful for evaluating the PM purification abilities of urban trees and forests in a precise, unbiased, and accurate manner.

The abilities of different tree species to remove PM from ambient air can be assessed through quantifying the mass of PM retained on leaf surfaces. To achieve this objective, the subtraction method1,2, the membrane filter method3,4,5, and the elution-weighing method coupled with particle size analysis6 have been applied to quantitatively estimate the mass of PM2.5 (diameter &#8804; 2.5 &#181;m), PM10 (diameter &#8804; 10 &#181;m) or total suspended particulate (TSP) retained on leaves. However, the accuracy of these methods basically depends on their performance in collecting PM retained on the leaf surfaces. At present, the conventional leaf cleaning method used in related studies often includes one or two steps, namely only water washing (soak and rinse leaves using deionized water)3,7 or plus brushing5,8,9. However, some studies10,11 have demonstrated that PM on leaf surfaces could not be completely eluted by the conventional cleaning method. As ultrasonic cleaning has the advantages of high speed, high quality, and little damage to the surface of the object, it has great potential to be used to collect the PM retained on leaf surfaces with complex microstructures. At present, ultrasonic cleaning has been applied in some studies to collect PM retained on leaf surfaces (i.e., put the leaves into deionized water, and use the ultrasonic cleaner to elute PM)12,13. However, this method is only used as a supplement to a leaf cleaning method, while it is not known whether the ultrasonic cleaning has a positive effect on collecting PM from leaf surfaces and its optimum operating parameters are also not clear. Our previous research has shown that the PM retained on Ginkgo biloba leaf surface could be completely eluted without destroying the leaf surfaces, if a proper ultrasonic cleaning procedure was supplemented to the conventional cleaning method11. However, the stability and general applicability of the ultrasonic cleaning parameters (ultrasonic power, time, and other information) to different plant species experiencing different dust retention periods are still not clear.
Currently, the mass of PM2.5, PM10, or TSP on unit leaf area has often been utilized to evaluate the abilities of different tree species to remove PM from ambient air14,15. Under the natural condition, the PM retained on leaf surfaces can be classified into two parts: the first part is the PM that can fall off leaves due to the effects of wind and rainfall, while the other part is the PM that is tightly adhered to leaf surfaces and cannot be easily washed off by rainfall. However, few studies have focused on the mass of both types of PM on leaf surfaces. In addition, the PM retention periods of leaves in different studies differ enormously. Thus, the comparability of the results of these studies will be poor, if the mass of PM retained on unit leaf area is adopted to assess the PM removal abilities of trees16. Consequently, the PM retention efficiency (the mass of PM retained on unit leaf area per unit time), as an alternative, was proposed to evaluate the PM purification effects of urban trees5,17. In general, there is still a lack of research in this aspect. It is extremely necessary to carry out relevant studies for different tree species to provide methodological basic and data support for assessing the PM removal abilities of different tree species accurately.
Here, three broadleaf tree species (G. biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) were selected to evaluate their PM removal abilities under two PM retention periods. The leaf sampling site was in Xitucheng Park (39.97&#176; N, 116.36&#176; E), located in an area with heavy pollution in Beijing. The three specific objectives of this study were: (1) to assess the efficiency of different leaf cleaning methods (water cleaning (WC), brush cleaning (BC), and ultrasonic cleaning (UC)) in eluting the PM on leaves, (2) to verify the effect of ultrasonic cleaning on eluting PM, and (3) to assess the retention efficiency of different tree species to PM1, PM2.5, PM5, PM10, and TSP.

<table><tbody><tr><td>MSA2258-1CE-DU ten-thousandth scale</td><td>Sartorius Scientific Instruments (Beijing) Co., Ltd.</td><td>MSA2258-1CE-DU</td><td>precision: 0.01 mg</td></tr><tr><td>The IS13320 laser granularity instrument</td><td>Beckman Coulter, Brea, USA</td><td>IS13320</td><td>working conditions: liquid/power samples; particle size range of measurement: 0.017-2000 &amp;mu;m</td></tr><tr><td>Epson Twain Pro high-quality scanner</td><td>Seiko Epson, Nagano, Japan</td><td>expression1680</td><td></td></tr><tr><td>Automatic image analysis software WinRHIZO</td><td>Regent Instruments Inc., Quebec, Canada</td><td>WinRHIZO Pro 2013a</td><td></td></tr></tbody></table>

assessing the particulate matter removal abilities of tree leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The PM retained on leaf surfaces had two types under natural conditions. The PM falls off easily by rainfall and wind under natural conditions is defined as the easily removable particulate matter (ERP). This type of PM was represented by the PM eluted by WC in this study. The PM that tightly adheres to leaf surfaces and cannot be easily washed off by BC and UC is defined as the difficult-to-remove particulate matter (DRP). This kind of PM cannot be eluted by natural rainfall and wind.
There was a significant difference among the five tree species in the mass proportions of various-sized PM eluted by different cleaning steps. The result showed that a large number of different sized PM were eluted from leaf surface by WC (Figure&#160;1, Figure 2, Figure 3, Figure 4, and Figure 5). The average eluted proportions (ERP) of various-sized PM of the five tree species were 31% and 35% under the SDR and LDR, respectively (Figure 6).
In addition, the WC showed a stronger effect on eluting PM on leaves of needleleaf tree species (P. tabuliformis and S. chinensis), especially for S. chinensis under the LDR period. As such, the elution percentage of WC was significantly higher than that of BC and UC (P &#60; 0.05) for all sized PM except PM&#62;10. After cleaning the leaves by BC, large fractions of various sized PM were also eluted, which were 28% and 29% under the SDR and LDR periods, respectively. Similar to WC, the most distinct elution effect of BC was observed for S. japonica. The elution percentage of BC was significantly higher than that of WC (P &#60; 0.05) for all fractions of PM under both the SDR and LDR periods. Moreover, the elution effect of BC was significantly higher than that of UC (P &#60; 0.05) except PM with diameter &#60; 5 &#181;m (Figure 6). Although a large fraction of PM could be eluted from leaf surface by WC + BC, some PM with smaller diameters still adhered on leaf surfaces. Subsequently, when UC was applied to clean leaves, the residual PM retained on leaf surfaces were eluted completely (Figure&#160;1, Figure 2, Figure 3, Figure 4, and Figure 5), and the elution proportions were 41% under SDR and 36% under LDR (Figure 6). In addition, the elution percentage of small sized PM was higher when the UC was applied. Consequently, the mass of PM would be underestimated apparently, if the conventional elution method was only adopted to elute the PM on leaves. Especially for S. babylonica, the average eluted proportions of PM in all diameter classes would be underestimated by 46%, which was higher than that for P. tabuliformis (43%), G. biloba (42%), S. japonica (31%), and S. chinensis (29%).
The AEleaf of different types of PM of the five tree species is shown in Table 1. There was a great difference in the retention efficiency calculated by two different methods. Compared with the result estimated by equation (2), the AEleaf calculated by equation (3): retention efficiency (mg/m2&#183;d-1) = the mass of the PM on a unit of leaf area (mg/m2) / the dust retention duration (d) was about 5 times higher. Especially for S. japonica, the PM1 of ERP calculated by equation (3) was 18.94 times higher than that calculated by equation (2). In this study, for ERP, the TSP AEleaf of different tree species varied between 12.69 and 34.69 mg&#183;m-2&#183;d-1 and decreased in the following order: P. tabuliformis &#62; S. babylonica &#62; G. biloba &#62; S. japonica &#62; S. chinensis. While in a previous study, the TSP AEleaf of different tree species varied between 35.27 and 85.79 mg&#183;m-2&#183;d-1 and decreased in the following order: S. japonica &#62; S. chinensis &#62; S. babylonica &#62; P. tabuliformis &#62; G. biloba. The retention efficiency of different tree species in retaining various sized PM of different types (ERP, DRP, TRP) could also vary. In this study, S. japonica exhibited the highest AEleaf in retaining the TRP of PM1 and PM2.5, which were 4.3 and 21.91 mg/m2&#183;d-1, respectively. S. babylonica had the highest AEleaf in retaining the TRP of PM5 (40.98 mg/m2&#183;d-1) and PM10 (62.01 mg/m2&#183;d-1). In addition, S. chinensis could retain more ERP of PM1, PM2.5, and PM5 than the other tree species.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58026/58026fig1.jpg" /> 
Figure 1: The particulate matter residue on leaves of Ginkgo biloba after different elution steps. A and B stand for the upper and lower sides of leaves of Ginkgo biloba. Different numbers stand for different elution steps. (1: without cleaning; 2: single water cleaning; 3: water cleaning + brush cleaning; 4. water cleaning + brush cleaning + ultrasonic cleaning). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig1large.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/58026/58026fig2.jpg" /> 
Figure 2: The particulate matter residue on leaves of Pinus tabuliformis after different elution steps. A and B stand for the concave and convex sides of leaves of Pinus tabuliformis. Different numbers stand for different elution steps. (1: without cleaning; 2: single water cleaning; 3: water cleaning + brush cleaning; 4. water cleaning + brush cleaning + ultrasonic cleaning). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig2large.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/58026/58026fig3.jpg" /> 
Figure 3: The particulate matter residue on leaves of Sophora japonica after different elution steps. A and B stand for the upper and lower sides of leaves of Sophora japonica. Different numbers stand for different elution steps. (1: without cleaning; 2: single water cleaning; 3: water cleaning + brush cleaning; 4. water cleaning + brush cleaning + ultrasonic cleaning). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig3large.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/58026/58026fig4.jpg" /> 
Figure 4: The particulate matter residue on leaves of Salix babylonica after different elution steps. A and B stand for the upper and lower sides of leaves of Salix babylonica. Different numbers stand for different elution steps. (1: without cleaning; 2: single water cleaning; 3: water cleaning + brush cleaning; 4. water cleaning + brush cleaning + ultrasonic cleaning). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig4large.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/58026/58026fig5.jpg" /> 
Figure 5: The particulate matter residue on leaves of Sabina chinensis after different elution steps. A and B stand for the conical and scale-form leaves of Sabina chinensis. Different numbers stand for different elution steps. (1: without cleaning; 2: single water cleaning; 3: water cleaning + brush cleaning; 4. water cleaning + brush cleaning + ultrasonic cleaning). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig5large.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/58026/58026fig6.jpg" /> 
Figure 6: The mass proportions of the various sized particulate matter retained on the leaves of different tree species. A and B stand for short (SDR) and long (LDR) dust retention periods, respectively. WC, BC, and UC stand for the single water cleaning, brush cleaning, and ultrasonic cleaning, respectively. Data are mean &#177; SE. Different letters (a, b, c) above the data bar indicate significant difference (P &#60; 0.05) among the different cleaning steps on the elution percentages of the different-sized particles under different dust retention periods, according to the Duncan test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58026/58026fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58026/58026table1.jpg" /> 
Table 1: The mass of different types of PM retained on unit leaf area. ERP, DRP, and TR stand for the easily removable dust retention capacity, the difficult-to-remove dust retention capacity, and the total dust retention capacity, respectively. Equation (2): retention efficiency (mg/m2&#183;d-1) = the subtract mass of the PM on unit leaf area under the LDR and SDR periods (mg/m2) / the dust retention duration between the LDR and SDR periods (d); Equation (3): retention efficiency (mg/m2&#183;d-1) = the mass of the PM on a unit of leaf area (mg/m2) / the dust retention duration (d).

Watch this Scientific Journal Video about Assessing the Particulate Matter Removal Abilities of Tree Leaves at JoVE.com

Assessing the Particulate Matter Removal Abilities of Tree Leaves

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

assessing-the-particulate-matter-removal-abilities-of-tree-leaves

Nanyang Technological University

Research

JoVE Journal

Environment

6.7K Views.  Beijing Forestry University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de relatie tussen stedelijke bossen en fijnstof vervuiling veld, zoals hoe boomsoorten te selecteren met een hoge deeltjes verwijdering vermogen. Het belangrijkste voordeel van deze techniek is dat de PM-verwijderingscapaciteiten van bomen nauwkeurig en kwantitatief kunnen worden beoordeeld door de PM die volledig op het bladoppervlak wordt bewaard, te verzamelen. Om te beginnen, selecteer vijf gezonde individuele bomen me...

Video: Assessing the Particulate Matter Removal Abilities of Tree Leaves

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

A Venturi Effect Can Help Cure Our Trees

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. According to these factors and their dynamics, small amounts of sap-compatible liquids (i.e. pesticides) can be injected into the xylem system, reaching their target from inside. This endotherapic method, called &#34;trunk injection&#34; or &#34;trunk infusion&#34; (depending on whether the user supplies an external pressure or not), confines the applied chemicals only within the target tree, thereby making it particularly useful in urban situations. The main factors limiting wider use of the traditional drilling methods are related to negative side effects of the holes that must be drilled around the trunk circumference in order to gain access to the xylem vessels beneath the bark.
The University of Padova (Italy) recently developed a manual, drill-free instrument with a small, perforated blade that enters the trunk by separating the woody fibers with minimal friction. Furthermore, the lenticular shaped blade reduces the vessels&#39; cross section, increasing sap velocity and allowing the natural uptake of an external liquid up to the leaves, when transpiration rate is substantial. Ports partially close soon after the removal of the blade due to the natural elasticity and turgidity of the plant tissues, and the cambial activity completes the healing process in few weeks.

Compared to the more traditional hole-based methods, most of which require the tree to be drilled, tools with lenticular blades transform the basics of endotherapy easing the closure of the wound and allowing the natural uptake of the solutions.

In woody plants, xylem sap moves upwards through the vessels due to a decreasing gradient of water potential from the groundwater to the foliage. ...

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemistry

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

A Method for Quantifying Foliage-Dwelling Arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...

Digital Leaf Physiognomy for Paleoclimate and Paleoecological Reconstruction

Reconstructing Terrestrial Paleoclimate and Paleoecology with Fossil Leaves Using Digital Leaf Physiognomy and Leaf Mass Per Area

Climate and environment strongly influence the size, shape, and toothiness (physiognomy) of plants&#39; leaves. These relationships, particularly in woody non-monocotyledonous angiosperms, have been used to develop leaf-based proxies for paleoclimate and paleoecology that have been applied to reconstruct ancient terrestrial ecosystems for the last ~120 million years of Earth&#39;s history. Additionally, given that these relationships have been documented in living plants, they are important for understanding aspects of plant evolution and how plants respond to climatic and environmental changes. To conduct these types of analyses on modern and fossil plants, leaf physiognomy must be measured accurately using a reproducible methodology. This protocol describes a computer-based method for measuring and analyzing a variety of leaf physiognomic variables in modern and fossil leaves. This method allows for the measurement of leaf physiognomic traits, in particular variables related to leaf serrations, leaf area, leaf dissection, and linearity that are used in the digital leaf physiognomy proxy for reconstructing paleoclimate, as well as petiole width and leaf area, which are used for reconstructing leaf mass per area, a paleoecological proxy. Because this digital leaf trait measurement method can be applied to fossil and living plants, it is not limited to applications related to reconstructing paleoclimate and paleoecology. It can also be used to explore leaf traits that may be informative for understanding the function of leaf morphology, leaf development, phylogenetic relationships of leaf traits, and plant evolution.

Author Spotlight: Leaf Trait Analysis for Climate and Ecology Reconstruction in Modern and Ancient Plant Communities

The protocol presented shows digital measurement and analysis of continuous leaf physiognomic traits on fossil leaves to reconstruct paleoclimate and paleoecology using the digital leaf physiognomy and leaf mass per area reconstruction methods.

Climate and environment strongly influence the size, shape, and toothiness (physiognomy) of plants&#39; leaves. These relationships, particularly in woody ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Biology

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Transpiration

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Comparing Transpiration Rates of Leaves
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Before beginning the experiments, gather leaves from four plant species that represent different ecological strategies, so that you have enough for one set of leaves per student. NOTE: If leaf-bearing plants are not available locally, an alternative is to grow the plant species of interest in the laboratory, or to purchase plants from a gardening store.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Next, cut lengths of rubber tubing to make roughly 50 cm sections and place one piece alongside a 5 mL pipette at each student workstation.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, set out a bucket or tray of water and ensure students have access to the appropriate clamps and stands.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out bottles of clear nail polish, as well as clear tape and clean microscope slides for the stomata exercise.
</div></li></ul></li></ol>

Assessing Transpiration Rates using Potometer - Prep

Comparing Transpiration Rates of Leaves
ExpandBefore beginning the experiments, gather leaves from four plant species that represent different ecological ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

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