Measuring Objects

SETTING THE SCALE

Fiji will recognize the meta data and automatically set the scale for most image files. If this is not the case, you can manually set the scale by:

  • Find the pixel size by going to Image > Show Info
    • You may need to use Plugins > Bio-Formats-Importer to import the metadata to properly display the pixel size info (e.g. when opening .tif files from the Hitachi TEM)
  • Analyze > Set Scale
  • Enter 1 into the “Distance in pixels”.
  • Input the pixel size into the “Known distance” field.
  • Enter nm or um into the “Unit of length” field, as appropriate.

If you don’t have metadata: you can set the scale by measuring the scale bar in the image (not as accurate as the method above).

  • Select the line tool. Click once at the beginning of the scale bar and then drag the mouse to the end point, and release.
  • Analyze > Set Scale
  • “Distance in pixels” should be automatically populated with the number of pixels in your line.
  • Input the known length of your scale bar into the “Known distance” field.
  • Enter nm or um into the “Unit of length” field, as appropriate.
  • Click the “Global” checkbox to apply the scale to all images that you open in the session.

ANALYSIS

Measuring length: To obtain measurements of specimen length.

  1. Select the line tool. Click once to specify the beginning point of your line and then drag the mouse to the end point, and release.
  2. Choose Analyze tab and select “Measure”. A new window will open. Scroll to the right to see “Length”

Measuring single area: To obtain area measurements of a single region of interest.

  1. Click on the appropriate area selection tool for your object and outline your ROI (region of interest).
  2. Choose the Analyze tab and select “Measure”. A new window will open. Scroll to the right to see “Area”.

Measuring the area of complex objects or multiple regions: To obtain area measurements for many ROIs or a complex object within an image.This method can also be used to count objects.

  1. Convert your image to grayscale by selecting Image > Type > 8-bit.
  2. Threshold the image by selecting Process > Binary > Make Binary. This creates a two-color (black and white) image based on a threshold of the intensities of pixels in the original image.
  3. If this mask does not look correct for your image, manually threshold your image by selecting Image > Adjust > Threshold. A histogram will open and masks will appear on your image. Adjust the slider below the histogram to change the pixel value cutoff until the mask selects all your pixels of interest. Select “Apply” and close the window.
  4. To count your objects, select Analyze > Analyze Particles. Once the Analyze Particles window opens, define your minimum and maximum size of your object (pixels), select “Show Outlines” and check “Display Results”, “Summarize” and OK.
  5. Additional windows will open with all data for each object, as well as summary data for all of the objects. This can all be copied and pasted into a spreadsheet.