Protocol

Authors

Christine Scoffoni, Lawren Sack

Overview

This protocol explains how to clear and image leaves and to measure key venation traits. Important vein traits include vein densities (length/area) and diameters for all vein orders, and the number of free-ending veins per area.

Background

This protocol outlines how to obtain the traits used to quantify structure and to relate to the functions of leaf venation architecture. Leaf venation architecture has numerous common functions across plant species—see Sack & Scoffoni, 2013 for review. Briefly, the leaf venation serves for mechanical support (Niklas, 1999), sugar and hormone transport in the phloem (Kehr & Buhtz, 2008), and, via the xylem, the replacement of water lost to transpiration when the stomata open for photosynthesis (Sack & Holbrook, 2006). However, venation architecture is highly diverse across species (Uhl & Mosbrugger, 1999; Roth-Nebelsick et al., 2001; Sack & Frole, 2006; Ellis et al., 2009; Brodribb et al., 2010). In dicotyledons, the leaf venation system typically consists of three orders of major veins and up to five higher orders of minor veins embedded in the mesophyll, with the vein orders arranged in a hierarchy; lower order veins are larger in diameter, with greater xylem conduit numbers and sizes, whereas higher order veins have greater length per leaf area (VLA, also known as “VLA”; Sack & Holbrook, 2006; McKown et al., 2010). Total leaf VLA has been shown to correlate with maximum hydraulic conductance and photosynthetic rate per area across species (Sack & Frole, 2006; Brodribb et al., 2007) and tends to be higher for species growing in high light. Major VLA has been found to play a role in determining the damage tolerance of the vein system, and in leaf drought tolerance (Sack et al., 2008; Scoffoni et al., 2011).

Materials/Equipment

Chemicals: Aqueous formaldehyde, glacial acetic acid solution, EtOH , NaOH, Bleach

Stains: Safranin, Fast green

Equipment: Flatbed scanner, light microscope, ImageJ software (National Institutes of health; free online, http://rsbweb.nih.gov/ij/)

Units, terms, definitions

Vein length per unit leaf area (VLA; mm mm^-2). Also commonly known as “VLA”. Can be measured for veins of each order, or summed for major or minor veins or the whole system (see below). Total VLA correlates well with the average distance between veins (a.k.a. “interveinal distance”; see Uhl & Mosbrugger, 1999), and with areoles per area.

Free vein endings per area = Number of free vein endings per unit area (mm^-2)

Major VLA = Sum of vein densities for 1°, 2° and 3° veins (mm mm^-2)

Minor VLA = Sum of vein densities for 4° veins and higher (mm mm^-2)

Vein diameter = typical cross-sectional diameter of a central vein segment for a vein of a given order (mm)

Procedure

Leaves can be preserved in formalin-acetic acid solution (37% aqueous formaldehyde solution, 50% ethanol, and 13% glacial acetic acid solution) until  they are ready for clearing.

LEAF CLEARING (simplified from Berlyn & Miksche, 1976)

• It may take several attempts for good results for given species. The method given below is flexible (variable times for given steps are presented) and so if an attempt fails, modify given steps as necessary for improved results. Keep notes on your attempts and you will eventually arrive at a functional process for leaves of given species. Enjoy!
• Chemically clear leaves using 5% NaOH in ethanol solution (let the leaf sit in it for a few hours to a few days depending on the species), or use a 2.5% solution for delicate tissues.
• When leaves appear transparent and softened, vacuum off the NaOH solution and rinse several times in H₂
• If a little color remains in the leaf, rinse out the H₂O and pour a 50% bleach solution on the leaf for 10-20s. Rinse twice with H₂O and let sit for at least 1 hour.
• Bring leaves into alcohol again using EtOH dilution series (30%, 50%, 70%, 100%). Each change needs no more than 5 mins. Vacuum off liquid.
• After the 100% EtOH stage, cover the leaf in 1% safranin (1g safranin/100ml of EtOH). The leaf should sit 2-30 minutes depending on the species. Gently rinse with 100% EtOH and vacuum off liquid.
• Cover leaf in 1% fast green (1g fast green/100ml of EtOH) and leave for a few seconds. Gently rinse with 100% EtOH and vacuum off liquid.
• Bring leaf back to water in the reverse dilution series. The leaf may look overstained, but once it is in water stage, the excess dye will come out and should leave dark-stained veins (see attached image). Skipping these steps and going straight to water can mean getting an overstained leaf or a damaged leaf – take care as your tissues are soft. Once in the H2O stage, if you find your leaf is understained, bring back to 100% EtOH and repeat the stain procedure.
• Once in the H₂O stage, leaves can be mounted in H₂O for vein imaging and analysis.

VEIN IMAGING

• The leaf is mounted with water in transparency film (CG5000; 3M Visual Systems Division)
• First, the leaf is scanned (flatbed scanner; Canon Scan Lide 90; 1,200 pixels per inch) to allow measurement of the density of 1^o, 2^o and 3^o veins, and the diameter of 1^o vein(s).
• Next, the leaf is imaged under the light microscope to allow measurement of higher order veins.
• Using a light microscope (DMRB; Leica Microsystems) with a 5× or 10× objective and digital camera (14.2 Color Mosaic; Diagnostic Instruments), image three rectangles (at least 1.5 mm²) centrally in the top, middle and bottom thirds of the leaf. A good practice is to make sure that the vein at the top of the image is part of a 2° or 3° vein depending on the species (see below), so you can orient yourself to the vein orders in the image later
• Image two areas focusing on a typical central 2° vein.
• To calculate the exact image magnification, resolution, scale and resolving power you are using, please refer to the calculator spreadsheet in Sack et al., 2014 (Supplemental Table 1).

QUANTIFICATION OF MAJOR VEINS AND WHOLE LEAF STRUCTURAL TRAITS USING SCAN LEAVES

Rules should be made for distinguishing vein orders in your leaves (for excellent advice, see Ellis et al., 2009). There is a level of subjectivity and uncertainty in making these distinctions among vein orders, so attempt to be rigorous and consistent, and keep notes on your rules for distinguishing vein orders (see image below for an example of vein order determination on Heteromeles arbutifolia). Minor veins should not be measured on scanned leaves given the lack of resolution. Below is the protocol we recommend:

• First, a few tips on how to use ImageJ :

1. Zooming in and out: zoom in on the scale by pressing the + button on your computer (you can zoom out using the -) OR in Image J by pressing the magnifying glass icon (10^th from the left) where left clicking will permit you to zoom in and right clicking will make you zoom out.
2. Always try to have the image taking up as much of your screen as possible.
3. Going up and down/ left and right within the image: when you have zoomed in and enlarged your image to fit most of your screen, the full image might not appear. You will have to move right and left and up and down depending on what you want to measure. To do so press the space bar key and hold it down. Notice a little hand appeared instead of your normal cursor. While keeping the space button pressed down, click on the mouse and keep it clicked as you move up and down or left and right.
4. Set your measurement before you start: go to Analyze, Set measurements and click the area and perimeter boxes, unclick all the others.

• Measuring leaf area and perimeter

1. Zoom out of the scale, and zoom in at leaf lamina insertion, where the lamina starts to come off the petiole (the more you zoom in the more precise your measurement will be).
2. Enlarge your image so that it takes up most of your screen.
3. Select the polygon icon (3^rd from the left) in Image j.
4. Double click where the lamina meets the petiole on one side of the leaf. You are going to be tracing around the leaf area. Follow the leaf margin with your cursor and click once to anchor a new point. The more points you anchor the more precise it will be. You can move up and down the image using as explained above the space bar key and mouse.
5. Once you have traced around the whole leaf, close off the area by double clicking where you started.
6. Press D and M to draw and measure
7. Copy the results from the results window that popped up. You want to report the area and perimeter.

• Measuring maximum leaf width

1. Using the straight line tool (right click again on line icon and choose straight line), trace a line more or less parallel to the leaf midrib, then drag it to one side of the leaf, with the line touching the margin furthest away from the leaf midrib. Draw by pressing D. Then drag the same line to the other part of the leaf, again choosing the furthest part away from the leaf midrib (use the zoom to be sure you are touching the leaf margin when drawing), and draw. It should look like the picture below (purple lines):                                                                                                                                                                                                                                                                                    Note how the purple line touches the leaf margin at its widest point in the leaf.
2. Then select the segmented line again by right clicking on the line icon and choosing segmented line. Draw a straight line between the two lines that you have just drawn (in purple on image above). The line (yellow line below) should be perpendicular to both purple lines.
3. Draw and measure by pressing D and M
4. In the results box, copy the length value of the leaf width, and paste in your spreadsheet.

• Measuring maximum leaf length

The maximum leaf length for most leaves will be equal to the leaf midrib. In that case, you can report the length of the leaf midrib as maximum leaf length.

However, some species might have lamina below the point of connection of the petiole and midrib, or above the midrib end. In that case, follow the same instructions as for measuring leaf width, but instead of drawing straight lines on both side of the leaf, draw lines at the top and bottom of the leaf lamina, so that the lines you draw touch the leaf tip and the lowest point of the leaf.

• Calculating leaf shape indices (accessory information, but can be importantly associated with venatin)

Two indices of leaf shape can be calculated from the traits described above : the length : width ratio and the perimeter ² : area ratio which is a size independent index of edge relative to size (Sack et al., 2003).

• Measuring midrib diameter

1. Zoom in at the middle of the midrib (at half the distance between the petiole/midrib connection and the leaf tip).
2. Select the segmented line by right clicking on the line icon and choosing segmented line.
3. Double click on one side of the midrib. You have anchored your point.
4. Now, draw to the other side of the midrib, keeping perpendicular to the midrib length and double click on the other end.
5. Your cursor is free from the line. Press D and M.
6. Your line is now colored. Check that it does follow well the diameter of the midrib, perpendicular to its length. Report value to your spreadsheet.
7. Measure the diameter again, and average both values onto the spreadsheet.

• Measuring midrib length

1. Zoom in on the bottom of the midrib (the point where the petiole connects to the midrib)
2. Select the segmented line by right clicking on the line icon and choosing segmented line.
3. Double click where the midrib begins (this would be at the level where lamina starts coming off the petiole). You have anchored your point.
4. Now, follow the midrib closely by clicking along the midrib. You want to draw over the midrib.
5. When the midrib ends, double click. Your cursor is free from the line. Press D and M.
6. Your line is now colored. Check that it does follow well the midrib. Report value to your spreadsheet.

• Rules to determine whether a leaf is palmately or pinnately veined.

A leaf is palmately veined if it has more than one 1o vein; otherwise, it is pinnately veined.

To be considered a 1° vein, the two following rules need to apply:

1) First order veins are those that depart from the petiole and go towards the margins.

2) Other 1o veins should be at least 75% of the diameter of the midrib. To check this, measure the thickness of the midrib at its middle (to account for vein tapering) by using the segmented line and the one of the vein you want to check and compare measurements. If the vein you measured has a diameter 75% or more of that of the midrib, then you can consider it a first order vein.

• Measuring other 1°vein diameters

1. Proceed as indicated in “Measure the midrib diameter” but instead of the midrib, do it for each one of the 1° veins. In an excel spreadsheet copy your results from all the diameters of your 1°veins (not including the midrib).
2. Report and average the values in your spreadsheet.

• Measuring other 1° vein length

1. Zoom in on the bottom of the midrib, at the lamina insertion.
2. Select the segmented line by right clicking on the line icon and choosing segmented line.
3. Double click where the 1° vein begins (this would be at the “insertion point”, i.e., where lamina starts coming off the petiole). You have anchored your point.
4. Now, follow the 1° vein closely by clicking along it. You want to draw over the vein.
5. When the 1° vein ends, double click. Your cursor is free from the line. Press D and M.
6. Your line is now colored. Check that it does follow well the vein.
7. Continue to the next 1° vein and repeat steps 1-6
8. Once all your 1° veins are drawn and measured, select all the measurements in your result section, copy, and paste onto an Excel spreadsheet.
9. Report and sum all the lengths together in your spreadsheet.

• Determining 2^nd vein orders

Before you start measuring, take a moment to look at your leaf and determine what type of 2° veins your leaf has:

• Leaves can have different types of 2° veins: they can be craspedodromous (large 2 veins branch straight from the midrib to the leaf margins), brochidodromous (large 2° veins loop one into the other, never touching the leaf margin), or eucampdodromous (large 2° veins eventually loop one into the other, but they do so really close to the margin, after having followed the margin for a little bit– eucampdodromous is sort of an intermediary type between craspedodromous and brochidodromous types leaves).
• Within these categories, we will distinguish three possible types of 2° veins, although these are not necessarily present all leaves: “large 2° veins”, “small 2° veins” and “other 2° veins”.

1. The large 2° veins are the most obvious ones. They branch off the midrib and go towards the margin. You can see those in uncleared leaves because they usually are thicker than other veins besides the midrib.
2. The small 2° veins also branch off the midrib. However, they usually are not as long, and may be indistinguishable from 3° veins half way or so toward the leaf margin. CAREFUL! Not all veins branching off the midrib are 2°. You can have 3° veins or even minor veins branching off the midrib. You will recognize small 2° veins because they will follow the same pattern as the large 2° veins, but will be perhaps thinner and will not reach the leaf margins. If you are hesitating as to whether a vein should be a small 2° or a 3°, look at the pattern of 3° and minor veins, which are smaller veins that form a regular mesh in the leaf. Do you think the vein in question is part of the 2° vein pattern that is distinct from the mesh made up of 3° or higher order veins?
3. Other 2° veins are veins that stand out as well, forming an obvious skeleton pattern, but that do not depart from the midrib. They usually are additions to the large 2° veins. See below for illustrations. But again, if in doubt, ask yourself if in that area, the vein in question is more continuous with the 2° vein pattern or with the mesh made up by the 3o and higher order veins.

• Measuring 2^nd vein length

1. Select the segmented tool. Double click on the first 2° vein, at the intersection of the midrib and 2° vein. Then click following that vein until the end. Double click once you have drawn over the whole vein. Press D and M.
2. The result box will show up with the length of that vein. Leave it opened, and go to the next vein. Repeat step 4 until all the 2° veins of the category (ex: large) are measured.
3. Once all veins are measured, select all the data in the result box (there should be one line per vein measured). Copy, and then paste it to a blank spreadsheet.
4. Enter and sum all the lengths together using the formula in excel: =SUM(data)
5. Copy the value, and 2^nd vein lengths in your spreadsheet.

• Measuring third order vein lengths

1. Partition the leaf into three parts: bottom third, middle and top third of the leaf. Select the Rectangular icon (the very first icon) and draw onto your leaf a rectangle making up half of the third of the whole leaf surface and on one side of the midrib, so as to avoid it (we have had it vary from 10 to 300mm², but depending on your leaf size, this could be smaller or larger than this range; make sure that the box spans at least two secondary veins). Draw your rectangle at the bottom of the leaf, avoiding if possible the midrib (although for very thin leaves that might be impossible), so that the rectangle fits in between the midrib and the leaf margin.
2. Press D and M, then drag your rectangle to the middle third of the leaf, place it like the first one between the midrib and margin and Press D.
3. Drag your rectangle toward the top third of the leaf; place it between the midrib and margin and Press D. (Note: some leaves are really narrow toward the top, and the rectangle you drew for the bottom and middle might not fit at the top. In that case, draw a new rectangle of more or less the same size, but that would be longer than wide, so that it fits on your leaf lamina. Be sure to measure the area of that new rectangle.)
4. Record the area of your rectangle (or rectangles) in your spreadsheet.
5. Measure the total length of the secondary veins if present by tracing over them using the segmented line. Press M and D. Once all are measured, copy the results onto a spreadsheet and sum them all up. Copy the summed up value, paste special “value” and copy the result in your spreadsheet.
6. Proceed to measure 3°veins by using the segmented line and tracing over all tertiary veins. Then repeat step 5 for the 3°veins results.
7. Repeat steps 5 and 6 for all your rectangles. We suggest using a different color for different vein orders.

• Calculating vein densities for major veins

1. Vein densities for each order are calculated as the ratio of vein length/leaf area for 1^o and 2^o veins, and as 3^o length / rectangle area for 3^o However, because your 3 vein length per area measurement might be biased by the amount of secondary veins included in the boxed area, we recommend measuring 3 ^o VLA as
2. Major VLA is calculated as the sum of the 1°, 2° and 3° vein densities

QUANTIFICATION OF VEINS FROM LIGHT MICROSCOPE IMAGES

• Measuring 2^nd vein diameter

From the light microscope images of 2° veins, measure the vein diameter centrally twice.

• Quantification of minor veins (see image below):

From each image at the top, middle, bottom part of the leaf, measure:

• Total image area
• Area occupied by 2° veins
• Area occupied by 3° veins
• 2° vein length
• 3° vein length
• Total length of 4° veins and higher
• Number of free vein endings
• Vein diameters centrally for two segments of each vein order (3°, 4°, 5°, 6° and 7° if existent)
• Minor VLA is calculated as
• If necessary, you can also remove the area occupied by 3^o veins from the denominator, but this might not be practical if there are many such veins in the image, and the area occupied in the image relatively very small.
• Total VLA can be calculated as . It would be best to calculate this for the image area without 1^o or 2^o Alternatively, total VLA can be calculated as major VLA plus minor VLA, or ideally as major VLA plus minor VLA × (1-fraction of leaf area occupied by major veins), where the fraction can be determined as the sum of diameter × VLA for 1^o, 2^o and 3^o veins. Values determined these ways tend to be extremely highly correlated.
• Free vein endings per area is calculated as

References

Berlyn GP, Miksche JP. 1976. Botanical microtechnique and cytochemistry. Ames, Iowa, USA: Iowa State University Press.

Brodribb TJ, Feild TS, Jordan GJ. 2007. Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology 144(4): 1890-1898.

Brodribb TJ, Feild TS, Sack L. 2010. Viewing leaf structure and evolution from a hydraulic perspective. Functional Plant Biology 37(6): 488-498.

Ellis B, Daly DC, Hickey LJ, Mitchell JD, Johnson KR, Wilf P, Wing SL. 2009. Manual of Leaf Architecture. Ithaca, NY: Cornell University Press.

Kehr J, Buhtz A. 2008. Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany 59(1): 85-92.

McKown AD, Cochard H, Sack L. 2010. Decoding leaf hydraulics with a spatially explicit model: principles of venation architecture and implications for its evolution. American Naturalist 175(4): 447-460.

Niklas KJ. 1999. A mechanical perspective on foliage leaf form and function. New Phytologist 143(1): 19-31.

Roth-Nebelsick A, Uhl D, Mosbrugger V, Kerp H. 2001. Evolution and function of leaf venation architecture: a review. Annals of Botany 87: 553-566.

Sack L, Caringella M, Scoffoni C, Mason C, Rawls M, Markesteijn L, Poorter L. 2014. Leaf vein length per area is not intrinsically scale dependent: avoiding measurement artifacts for accuracy and precision. Plant Physiology 166(2): 829-838.

Sack L, Cowan PD, Jaikumar N, Holbrook NM. 2003. The ‘hydrology’ of leaves: co-ordination of structure and function in temperate woody species. Plant Cell and Environment 26(8): 1343-1356.

Sack L, Dietrich EM, Streeter CM, Sanchez-Gomez D, Holbrook NM. 2008. Leaf palmate venation and vascular redundancy confer tolerance of hydraulic disruption. Proceedings of the National Academy of Sciences of the United States of America 105(5): 1567-1572.

Sack L, Frole K. 2006. Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Ecology 87(2): 483-491.

Sack L, Holbrook NM. 2006. Leaf hydraulics. Annual Review of Plant Biology 57: 361-381.

Sack L, Scoffoni C. 2013. Leaf venation: structure, function, development, evolution, ecology and applications in past, present and future. New Phytologist 198: 938-1000.

Scoffoni C, Rawls M, McKown AD, Cochard H, Sack L. 2011. Decline of leaf hydraulic conductance with dehydration: relationship to leaf size and venation architecture. Plant Physiology: in press.

Uhl D, Mosbrugger V. 1999. Leaf venation density as a climate and environmental proxy: a critical review and new data. Palaeogeography Palaeoclimatology Palaeoecology 149(1-4): 15-26.