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Quantifying leaf vein traits

Christine Scoffoni, Lawren Sack
Contributors : LawrenSack1762 points  DS Chatelet101 points  Christine Scoffoni, Lawren Sack

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This protocol explains how to clear and image leaves and to make basic measurement of 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.


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 common functions across plant species, serving for mechanical support (Niklas, 1999), sugar and hormone transport in the phloem (Kehr and Buhtz, 2008), and, via the xylem, the replacement of water lost to transpiration during photosynthesis (Sack and Holbrook, 2006). However, venation architecture is highly diverse across species (Uhl and Mosbrugger, 1999; Roth-Nebelsick et al., 2001; Sack and 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 area (vein density; Sack and Holbrook, 2006; McKown et al., 2010). Total leaf vein density has been shown to correlate with maximum hydraulic conductance and photosynthetic rate per area across species (Sack and Frole, 2006; Brodribb et al., 2007) and tends to be higher for species growing in high light. Major vein density 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).


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,

Units, terms, definitions

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

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

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

Minor vein densities = 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)


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 and 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 aqueous 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 H2O.
  • 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 100% 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 100% 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.


  • The leaf is mounted with water in transparency film (CG5000; 3M Visual Systems Division)
  • 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, 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).
  • First, the leaf is scanned to allow measurement of the density of 1o, 2o and 3o veins, and the diameter of 1o vein(s). Next, the leaf is imaged under the light microscope to allow measurement of higher order veins.
  • The whole leaf is scanned (flatbed scanner; Canon Scan Lide 90; 1,200 pixels per inch)
  • 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.


Quantification of whole leaf features:

  • Using ImageJ software (National Institutes of Health) measure leaf area, perimeter, length and width.
  • Two indices of leaf shape can be calculated. The length:width ratio and the perimeter²:area ratio which is a size-independent index of edge relative to size (Sack et al. 2003).


Quantification of major veins (see image below):

  • Using ImageJ software, measure the total lengths of 1° and 2° veins.
  • 3° vein lengths are measured in three rectangles per leaf size (area of rectangle would depend on leaf size) located centrally in the top, middle and bottom thirds of half the leaf.
  • Measure the 1°vein diameter, excluding the bundle sheath, by averaging two measurements done centrally in the 1°vein.
  • Vein densities for each order are calculated as the ratio of vein length/leaf area for 1o and 2o veins, and as 3o length / rectangle area for 3o veins.
  • Major vein density is calculated as the sum of the 1°, 2° and 3° vein densities
  • 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 vein density is calculated as 

(the sum of the vein lengths of 4º veins and higher) / (total image area - area occupied by 2º veins)

  • If necessary, you can also remove the area occupied by 3o 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 vein density can be calculated as

(the sum of the vein lengths of all orders of veins) / (total image area) 

It would be best to calculate this for the image area without 1o or 2o veins. Alternatively, total vein density can be calculated as major vein density plus minor vein density, or ideally as major vein density plus minor vein density × (1-fraction of leaf area occupied by major veins), where the fraction can be determined as the sum of diameter × vein density for 1o, 2o and 3o veins. Values determined these ways tend to be extremely highly correlated.

  • Free vein endings per area is calculated as

(the number of free vein endings) / (total image area - 2º  vein area)




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

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

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

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

Kehr J, Buhtz A (2008) Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany 59, 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, 447-460

Niklas KJ (1999) A mechanical perspective on foliage leaf form and function. New Phytologist 143, 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, 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, 1567-1572

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

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

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, 15-26


Contributors to this page: Admin26202 points  , LawrenSack1762 points  , Admin36802 points  , DS Chatelet101 points  and Christine Scoffoni, Lawren Sack .
Page last modified on Tuesday 06 of August, 2013 21:53:54 EST by Admin26202 points . (Version 15)