Fig. 2.

Appearance of images at each stage of the processing and analysis with ImageJ. Major steps are: (A) unprocessed image: JPEG format, image is coloured; (B) background subtraction: dark background, light-coloured objects; (C) 8-bit adjustment: inverted image, white background with black objects; (D) threshold adjustment: set contrast to define objects under the red-scale setting; and (E) analysis: Count particles with size 10 pixels² to 100 pixels² and circularity 0·5–1·0 (Carduus-specific settings). Scale bar = 100 pixels.