Quantifying fine root and leaf morphology (and seeds) from desktop scans

Protocol

Author

Louise Comas

Overview

Morphology of ephemeral tissues is linked to tissue functioning such that suites of traits in both leaf and root tissues have been associated with plant growth strategies (Reich et al., 1999; Comas et al., 2002; Comas & Eissenstat, 2009). This protocol outlines the use of a desktop scanner and commercial software to image and analyze leaves and roots. It has the advantage of using the same hardware and software for both leaves and roots, efficiently analyzes large numbers of samples with greater accuracy than some equipment specifically designed for these purposes, and can also be used to quantify the number and size of seeds. This protocol is particularly useful for fine roots, as the software used here calculates a range of root-specific measures of architecture. If you do not have access to the required software you may like to see an alternative method using freeware: Measuring leaf perimeter and leaf area.

Background

Desktop scans can be used to analyze relatively small samples compared to other equipment (e.g. Comair Rootscanner; LI-COR leaf scanner). Representative sub-samples can be used to quantify larger samples if needed. This can be done using the relationship between sub-sample morphology and dry weight, and the dry weight of the larger sample. For leaves, use of desktop scanners and imaging is faster and more accurate than leaf scanners (O’Neal et al., 2002) and can easily measure small and needle-like leaves. For roots, these methods are an improvement over the accuracy of equipment such as the Comair Rootscanner, which is insensitive to thin roots that comprise substantial length in a sample (Kato et al., 2010). The approach can also be used to quantify a greater number of morphological measures such as root diameterm and architecture.

Materials/Equipment

  • a PC computer
  • desktop scanner with transparency adapter and software that allows for the capture of black/white line art images in transparency mode with automated brightness and/or contrast settings
  • water-tight acrylic trays made to fit transparency adapter and hold roots in water (~1.5 cm deep)
  • tweezers, plus Teflon-coated or plastic tweezers
  • photoshop
  • Command prompt (a utility for the PC)
  • Delta-t scan (note: WinRhizo, which is more costly, can be used with similar accuracy (Bouma et al. 2000))

Units, terms, definitions

SRL = specific root length, root length per dry mass, m g-1average root diameter = mean root diameter of the sample modal root diameter = most frequent root diameter of the sample root tissue density = specific tissue density, dry mass per volume, g cm-3branching frequency*= root tips per length, tips m-1

SLA = Specific leaf area, leaf area per dry mass, cm g-1*this is a course measure of root architecture, meaningful if scans are limited to two root orders.

*this is a course measure of root architecture, meaningful if scans are limited to two root orders

Procedure

Scanning roots

  1. Make sure the top light source is installed and the scanner is ON BEFORE the computer is turned on. Otherwise you will have to reboot.
  2. Open Adobe Photoshop 6.0

    In Photoshop, under the File menu click on Import and choose the software for your scanner.

  3. In the Scanner Control Window:

    Set Scanner option to Transmissive

    Set Scan mode to Lineart, B/W (1 bit)

    Set resolution to 600 DPI (minimum dpi for reliable diameter measurements of fine rooted species)

    Set the brightness to automatic.

    In the Preview Window:

    Make sure the small icon of a portrait (along the left window edge) is selected as opposed to the negative image of the portrait.

    If you need to resize the preview area (the red square overlaid on the image of the scanner window is the area that will be scanned for preview), you can select another area under Settings then Preview area. A preview area larger than you need will just take a lot more time.

  4. Put the tray on the scanner and fill with just enough water to cover the bottom. If any air bubbles appear on the tray surface, try tapping them out or setting water out in a flask over night to de-gas. Spread the roots out on the tray close to the front edge while leaving about 1 cm margin. Use only plastic or Teflon (green) coated tweezers and be very careful not to scratch the trays. Arrange the roots so there is minimal crisscrossing of roots and the roots are distributed randomly (ie do not line them up) in a small square or rectangular area – you will have to draw a box around them later. You may have to cut up some of the root pieces to more easily spread them out but if you are interested in architectural measures be careful how you do this. If you have a very large sample, scan a representative subsample and dry this subsample separately to back-calculate root length measures of the total sample. This will save time and computer memory.
  5. Once the roots are arranged, close the scanner lid. If the transparency adapter for the scanner is a moving light source, make sure its calibration window (the lighted strip along the front of the scanning window) is clean and unobstructed.
  6. Click on Preview in the Preview Window. Use the mouse to move and size the area to be scanned around the sample (typically a dotted box). Occasionally the automatic setting for brightness over-exposes and whites everything out. If this happens, readjust the dotted square – if this readjustment doesn’t solve the problem, manually select the brightness in range of the other values the auto-setting has been in (usually from -35 to +15 in our system).
  7. If you have trouble visualising the roots because they are very fine or very pale, consider staining them with a dye like neutral red or analine blue to increase the contrast (see notes and troubleshooting below).
  8. Choose Scan. After the scanning is complete, click Exit (not the box in upper right corner) to close screen and access the picture. Save the picture of the sample in TIFF (.tif) format. Make the first letter of the file name is the same for all samples and choose a letter that will not be used in any of the file names or extensions (i.e. use letters like X, Z) – this is important for batch processing the images later. Edit images as needed in Photoshop. Analyze a sample before scanning too many samples to make sure that images will be accepted by the analysis software, especially if the scanner software is updated.

Scanning leaves

  1. Make sure the top light source is installed and the scanner is ON BEFORE the computer is turned on. Otherwise you will have to reboot.
  2. Open Adobe Photoshop 6.0

    In Photoshop, under the File menu click on Import and choose the software for your scanner.

  3. In the Scanner Control Window:

    Set Scanner option to Reflective (Flatbed)

    Set Scan mode to Lineart, B/W (1 bit)

    Set resolution to 300 DPI

    Set the brightness to automatic.

    In the Preview Window:

    Make sure the small icon of a portrait (along the left window edge) is selected as opposed to the negative image of the portrait

    If you need to resize the preview area (the red square overlaid on the image of the scanner window is the area that will be scanned for preview), you can select another area under Settings then Preview area. A preview area larger than you need will just take a lot more time.

  4. Lay the leaves out on the glass close to the front edge while leaving about 1 cm margin. Arrange the leaves in a square or rectangular area – you will have to draw a box around them later. Make sure there are no overlapping leaves. You can cut some of the leaves if you have species with large leaves. If you have to use tweezers to help move the leaves, use only plastic or Teflon (green) coated tweezers and be very careful not to scratch the glass on the scanner.
  5. Place a white sheet of paper on top of your leaves and close the scanner lid. If the transparency adapter for the scanner is a moving light source, make sure its calibration window (the lighted strip along the front of the scanning window) is clean and unobstructed.
  6. Click on Preview in the Preview Window. Use the mouse to move and size the area to be scanned around the sample (typically a dotted box). Occasionally the automatic setting for brightness over-exposes and whites everything out. If this happens, readjust the dotted square – if this readjustment doesn’t solve the problem, manually select the brightness in range of the other values the auto-setting has been in (usually from -15 to +30 in our system).
  7. Choose Scan. After the scanning is complete, click Exit (not the box in upper right corner) to close screen and to access the picture.
  8. Save the picture of the sample in TIFF (.tif) format. Make the first letter of the file name is the same for all samples and choose a letter that will not be used in any of the file names or extensions (i.e. use letters like X, Z) – this is important for batch processing the images later. Edit images as needed in Photoshop. Analyze a sample before scanning too many samples to make sure that images will be accepted by the analysis software, especially if the scanner software is updated.

Analyzing images

Note: Commands inside are what you type on the screen. These directions involve creating a Dos -run’ file and analyzing the image in batch mode.

  1. Open Command Prompt. It is located in the start menu, at the top of the menu.
  2. Get into the directory that the image files are in. If your images are in the C drive: { c: } then { cd/ } + the directory that the image files are in, e.g. { cd Summerfieldexp }
  3. Next create a file that lists your file names: { dir /o:n /b> list } This creates a file called -list’ which is a list of all the file names in that directory.
  4. { edit list } (to get into the editor)

    delete -list’ from the list and any other files not to be run

  5. In the Search menu go to Change.

    In the find box enter the 1st letter the file names (ie X, Z)

    In the change box enter Image File Name {12 spaces} : {the pathname to the files}{1st letter of the file name}

    For example you might enter Image File Name{12 spaces}:c: Summerfieldexpz

    Be aware that Delta-T scan may have problems if there are spaces in any names of files or folders so you may need to remove these spaces.

  6. Save the file twice using the Save as command. The First save in folder where you have the images (so you have a record of if with your files). Then save in C drive DT Scan folder because to run batch files in DT scan, the run file has to be in the same folder as the program. Outputs, however, can be sent to a different folder.
  7. Exit.
  8. Go into C drive, dt-scan and edit an existing run file already set up for leaf or root analysis ( a sample copy is provided by Dt-scan)

    c:

    cddtscan

    edit roots1.run

  9. For all analyses, lines to change:
  • output destination, change output file name and path but leave extension as it is (.plt)
  • check that the image background color is set to the background color of your images (should be white unless different settings or programs were used to acquire images )
  • erase old file names (those will be at bottom of run program)
    For root analyses:
  • check analysis type and change depending on your preference (options include root length, length sin length ℼ /2 plus others described in Dt-scan manual. (use alt + numpad 227 to insert in text file; use alt + numpad 233 for ℼ; note: this code may change depending on software versions and platforms) To analyze branching frequency, you will need tip counts as well as object counts (used to subtract the starting point of each root cluster, which is otherwise counted as a tip).
    NOTE: For root diameter, use Sin method for more accurate diameter values. If have a mix of root sizes, choose ℼ /2 method to more precisely find modal diameter peaks in the root distribution but the values will be underestimated).
    For leaf analyses, lines to change:
  • make sure analysis type is set to area

10. Save as your run file name (e.g. myroots.run) and Exit.

11. Add rootlist to run file { copy myroots.run + list }

Then { edit myroots.run } to check that all the file names follow the lines of the program without having an empty space and the semicolons should also line up. Save any changes and exit.

12. At the ” C:dtscan> ” prompt, enter { dt-scan /Bmyroots }

You should see DT open and start scanning images. This should take while and you should see dt-scan open each image. If it doesn’t, check the output files. The pathnames to your images may have errors. If so, your output file will give an error message for each file. Otherwise there may be problems with the pathname to the output file.

13. View results. There are two output files with the same file names; one has the extension .out and the other, .plt. The .out file is the long version of the output in which all the measured values are given with labels. The .plt file is formatted for direct input to a spread sheet; only some values are given and no columns are labeled (you can confirm the column labels with the .out file). Output files can be opened using edit in Command Prompt, MS Word or Excel.

Notes and troubleshooting tips

Clean roots can stay refrigerated about 1 week in water if time is needed for scanning. If more time is needed to scan roots, a crystal or two of thymol (C10H14O) can be added to each sample stored in distilled water. Thymol, however, smells and should be used in a fume hood with gloves and proper disposal. Ethanol (EtOH) may be used in various concentrations but with caution because it can cause shrinkage of tissues.

Neutral red (0.16 g/L) is recommended for staining roots digitized for morphological measures to insure good contrast if automated brightness settings are not available (Bouma et al., 2000). This stain can also act as a preservative if more time is needed to process scans (up to about 3-4 weeks). EtOH can remove some of this stain so that mycorrhizal colonization can be assessed following analysis for morphology but EtOH does not remove the stain completely so this root material will be difficult to assess for colonization. To insure high quality scans of the material, software that has automated settings for brightness and contrast can be used in lieu of staining the roots for contrast.

Links to resources and suppliers

Delta-t scan software: http://www.delta-t.co.uk

Literature references

Bouma TJ, Nielsen KL, Koutstaal B. 2000. Sample preparation and scanning protocol for computerised analysis of root length and diameter. Plant and Soil 218(1-2): 185-196.

Comas LH, Bouma TJ, Eissenstat DM. 2002. Linking root traits to potential growth rate in six temperate tree species. Oecologia 132(1): 34-43.

Comas LH, Eissenstat DM. 2009. Patterns in root trait variation among 25 co-existing North American forest species. New Phytologist 182(4): 919-928.

Kato Y, Okami M, Tajima R, Fujita D, Kobayashi N. 2010. Root response to aerobic conditions in rice, estimated by Comair root length scanner and scanner-based image analysis. Field Crops Research 118(2): 194-198.

O’Neal ME, Landis DA, Isaacs R. 2002. An inexpensive, accurate method for measuring leaf area and defoliation through digital image analysis. Journal of Economic Entomology 95(6): 1190-1194.

Reich PB, Ellsworth DS, Walters MB, Vose JM, Gresham C, Volin JC, Bowman WD. 1999. Generality of leaf trait relationships: A test across six biomes. Ecology 80(6): 1955-1969.

Leave a Reply