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Plant Detectives Manual: a research-led approach for teaching plant science

Appendix E: Software tips

E.1) Scanalyzer software output and data interpretation

The following concepts and definitions are adapted from the LemnaLauncher and LemnaMiner Manual (v. 10.03.08) from Lemnatec.

Area

Area is the number of pixels there are in an object. Note that areas with different colours can be estimated. These colours relate to differences in pigmentation produced by genotypic difference, developmental stage or growing conditions. Most quantitative measurements of size are based on area measurements. Based on these values and colour classification, objects can be characterised to identify colour distributions, or area can be related to biomass or movement of organisms.

Compactness

Compactness is defined as the ratio between the area of the object (‘ObjectArea’) and the area of the ConvexHullArea (the smallest geometrical object without concave parts that covers the whole object). In simpler terms, compactness provides an important quantitative value describing the subjective visual assessment of being compact.

MinEnclosing Circle diameter

This value describes the radius of the minimum hole the object would be able to pass through. This radius refers to the manually less stringently definable maximum ‘diameter’ that is often measured with a caliper. It is particularly suitable for asymmetrical objects that differ greatly from circles.

Eccentricity

As symmetry and regular growth are common in nature, the extent of non-centric shape can describe important effects; e.g., deviations from the regular growth scheme. Values range from 0 (corresponding to a circle) to 1 for a line.

 

Eccentricity = sqr (μ20 –μ02) + 4 * sqr (μ11)/sqr (μ20 + μ02)

 

Below is a grid representation showing the columns and row of a 24 cell grid. Note that the red tape on the top right of your tray should help you to identify what genotype is in each cell.

 

Image

E.2) Photoshop

This brief ‘how to’ for optimising images in Photoshop is useful for editing scanned images, such as of MS agar plates. There are three steps in optimising your root scan: auto levels, manual adjustment and sharpening.

 

Auto levels: Selecting this will automatically adjust the black and white point of your image, making black parts darker and white points lighter:

  1. select the roots and the area around them, avoid the white label
  2. go to Image: Adjustments: Auto levels

OR, use the keyboard shortcut: CTRL+SHIFT+L.Your roots should suddenly look a lot clearer.

 

Manual adjustment: This is a good way to make your roots clearer, and a backup option in case there is limited improvement by selecting auto levels:

  1. select the roots and the area around them, avoid the white label
  2. go to Image: Adjustments: Levels
  3. Select OK when the roots become most clear

 

Sharpening: If there are fuzzy edges on your scan, sharpening the image can make the roots easier to see. This step is optional, and usually unnecessary:

  1. select the entire image (CTRL+A)
  2. Go to Filter: Sharpen: Sharpen

You can repeat this if the first sharpening doesn’t work, but keep in mind that the more you sharpen your image, the more pixelated it will appear.

 

E.2) ImageJ

ImageJ, a useful image-processing application that is freely available for download, can be used for root length analysis. Click here to download ImageJ.

There is an online manual available from the same link and an abundance of user-generated ‘how-to’ forums can be found by ‘googling’ your query. It is an excellent tool for taking post-hoc measurements, such as root length or leaf area, and is simple to use. The following instructions on how to measure root length can also be used for taking leaf area or other relevant measurements.

  1. Begin by taking an image. The best images of plants grown on plates are obtained using a flatbed scanner with the agar face (not the lid) of the sealed plate facing down on the glass. Include a ruler as a scale and use a piece of black cardboard for the background. ImageJ can be used with most image formats (e.g., jpeg or tiff).

Open ImageJ. To open an image file, click File: Open. Select your image.

Set the scale.

  1. Use the line tool to draw a 1-centimetre (cm) line, using the ruler in your image as a guide. Press and hold the ‘shift’ key for a straight line.

TIP: zoom by using the zoom tool on the tool bar, or simply hover the mouse over the area you wish to zoom on and press the ‘+’ or ‘–’ key.

  1. Click on Analyse: Set scale. In the dialogue box, enter 1 in the known distance box and change the unit of length to cm. Tick the global box — this applies the scale to all of the images you open in this session. If you have different scales for different images, set the scale first! Notice that the distance in pixels box will automatically change based on the known distance.
  1.  
  2. Now you are ready to trace and measure the roots.
  1. Right click the line tool icon and change the setting to segmented line. This allows you to trace the root.
  2. Trace the root, clicking when you need to change the direction of the line. When you get to the end of the root, double click to stop drawing.

TIP: adjusting the brightness/contrast of the image can make it easier to see the roots. To do this, Click Image: Adjust: Brightness/Contrast. In the pop-up window, click Auto. This usually gives good results, but if you are not happy, you can manually adjust the levels.

  1. Click Analyze: Measure. In the Results window, review the length parameter. If you have set the scale correctly, this will be the length of the root in cm. You can only trace one root at a time, so be sure to click measure after each root. The Results window will hold all of the measurements from one image and can be saved and/or copied into Excel if desired.

TIP: explore the other tools in ImageJ, such as the polygon tool for leaf area measurements or the angle tool for quantifying gravitropic response.

E.2) EZ-Rhizo

After downloading EZ-Rhizo, refer to the website for a tutorial, video demonstration and a forum for user feedback. Click here to download EZ-Rhizo.


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