Measuring ''A. thaliana'' Phenotype using FIJI by Hand

Protocol: Analyzing Sample Area with ImageJ. Written by Rina Rosnow, edited and updated by Hanae Togami, and edited by Hope Ebert (Photos of A. thaliana and related results added by Hope).

1. Open ImageJ
   1. Open image file
   2. Go to file > open
   3. Select desired image
2. Set scale
   1. Zoom into scale bar on the original image
   2. Select ‘linear selecting tool’ on scale bar
   3. Draw a line along the length of the scale bar on the image by clicking and dragging
   4. Go to analyze > set scale
      1. Enter known distance and units
      2. If the scale bar is the same length in all of your images, check ‘global’ to apply scale bar length to all images
      3. Click ‘okay’
3. Set measurements
   1. Go to analyze > set measurements
   2. Select ‘area’
   3. Select ‘limit to threshold’
   4. Click ‘okay’
4. Adjust brightness and contrast
   1. Go to image > adjust > brightness/contrast (or ctrl+shift+c)
   2. Use slide bars to adjust image to desired appearance (you want the background as dark as possible without obscuring the edges of the sample)
   3. Click ‘apply’
5. Make image binary
   1. Go to image > type
   2. Select ‘8 bit’
   3. Go to process > binary
   4. Select ‘make binary’
6. Adjust threshold
   1. Go to image > adjust > threshold (or ctrl+shift+t)
   2. Select Over/Under in dropdown menu and check ‘dark background’
   3. Use slide bars to select images/remove background
      1. Put upper limit to max (bottom slide bar)
      2. Adjust lower limit using top slide bar
   4. Click ‘apply’
7. Measure image
   1. Select ‘wand’ tool in tool bar
   2. Click anywhere inside the sample to select the sample
   3. Go to analyze > measure (or ctrl+m)
8. Record measurements in the master log
9. Save ImageJ image in dated folder
   1. Select file > save as
   2. Select ‘tiff’
   3. Save image as “GenX_mmddyy_GrowBoxID”