Pattern analysis and acuity control require all images be scaled to a uniform number of pixels per unit length – a number that will vary with every study. Photographing different samples from slightly different distances can be controlled for with a scale bar, nevertheless we recommend taking photographs at uniform distances from the subject whenever possible. Particular care should be taken to photograph different treatments at the same distances for unbiased pattern analysis. Position the scale bar level with the sample and photograph from overhead if possible. If photographing from an angle rather than directly overhead a horizontal circular disk could be used as a scale bar (the disk’s maximum width will always equal its diameter whatever angle it’s viewed from) or place a straight scale bar/ruler side-on to the camera. Even if your initial hypothesis does not concern pattern, size or distance it is always good practice to include a scale bar.
Measuring the Scale Bar
Once you have generated/loaded an mspec image, simply select the line-selection tool in ImageJ, draw a line along the scale bar in the image and press “S” on your keyboard. A dialog box will ask you to specify how long the selected length is. The units are arbitrary, however the same unit must be used throughout the entire image processing stream. e.g. if you use millimetres then when specifying a viewing distance for acuity control that distance must also be in millimetres. We recommend using millimetres for all scales. Once you have created the scale bar an ROI is created which stores the relevant information, then press “0” (zero) on your keyboard to save the scale bar ROI to the mspec image.