Table I. Definitions of terms relating to image analysis and their application to the estimation of VLA from images (Ruzin, 1999).

Note that image magnification, resolution of image, scale of image, and resolving power are interrelated (Supplemental Table S1). Image quality for measuring VLA depends on magnification and resolution being sufficient to discern all veins and, additionally, on sufficient leaf clearing and staining and contrast in the image.

Term	Definition	How to Quantify for Measurement of VLA
Accuracy of estimate	Degree of conformity of a measured or calculated quantity to its actual (true) value	How close are the values to the true value? This is determined using digital tracing in high-magnification, high-quality images in which all veins can be observed sharply
Precision of estimate	Degree to which several measurements provide answers very close to each other	Coefficient of variation across experimenters
Image quality	Perceived image degradation (compared with an ideal or perfect image); this is affected by many factors, including especially sharpness, the amount of detail the image can convey	Are all the veins discernible? Low image quality may arise due to too-low magnification and/or too-low resolution, and/or blurring of the image caused by downsampling, and/or not sampling the vein network representatively (e.g. zooming in within a single areole or including large major veins in an image of minor venation)
Microscope objective magnification	Magnification contributed by the objective of the microscope: one element in the total image magnification	Given on the microscope objective
Image magnification	Ratio of the apparent size of an object in the image to its true size	Actual size of the digital image, typically embedded in the image file, relative to its actual size; because the camera magnifies the image beyond the microscope, this can be many times larger than the microscope objective magnification
Total display magnification	Ratio of the apparent size of an object of a displayed image including its zoom to its true size	Size of the image when displayed on a monitor at 100% size or with further zooming can be quantified using a ruler on the screen relative to its actual size; typically, this is many times larger than the microscope objective magnification, as it depends on image magnification (i.e. in the image file) and additionally on the computer operating system and/or software display resolution settings and/or the display monitor
Resolution of image	Ratio (pixels mm−1) of the image length in picture elements (pixels) to the length of the image (mm)	Dividing the length of the image in pixels by its actual length
Scale of image	Ratio (pixels mm−1) of the image length in picture elements (pixels) to the length of the field of view (mm)	Dividing the length of the image, or a ruler or micrometer in the image in pixels by its actual length; the scale of image equals the product of the image magnification and the resolution of the image
Resolving power	Ability to distinguish two points that are close together, and the ability to resolve minor veins in the image	Resolving power increases with the scale of the image; the diameter of the smallest vein should be larger than three pixels at minimum