Perceptually accurate display of two greyscale images as a single colour image

Summary Life scientists often desire to display the signal from two different molecular probes as a single colour image, so as to convey information about the probes’ relative concentrations as well as their spatial corelationship. Traditionally, such colour images are created through a merge display, where each greyscale signal is assigned to different channels of an RGB colour image. However, human perception of colour and greyscale intensity is not equivalent. Thus, a merged image display conveys to the typical viewer only a subset of the absolute and relative intensity information present in and between two greyscale images. The Commission Internationale de l'Eclairage L*a*b* colour space (CIELAB) has been designed to specify colours according to the perceptually defined quantities of hue (perceived colour) and luminosity (perceived brightness). Here, we use the CIELAB colour space to encode two dimensions of information about two greyscale images within these two perceptual dimensions of a single colour image. We term our method a Perceptually Uniform Projection display and show using biological image examples how these displays convey more information about two greyscale signals than comparable RGB colour space‐based techniques. Lay description It is often useful to display the fluorescent signal from two different molecular probes as a single colour image so that information about the probes’ relative concentrations as well as their spatial corelationship can be visualized. A common way to accomplish this effect is to assign each greyscale signal to a different channel of an RGB colour image. However, this method fails to convey much of the information about the two greyscale images, because humans’ perception of colour and monochrome values is not equivalent. We have used the Commission Internationale de l'Eclairage L*a*b* colour space (CIELAB) to display two greyscale images as a single colour image. Our method encodes two dimensions information about the greyscale images as two, perceptually independent and uniform quantities – hue and luminosity – in the colour image. Our technique, which we termed a perceptually uniform projection display, conveys more information about two greyscale signals than comparable RGB colour space‐based techniques. We apply this method to display several types of information commonly encountered in cell biology images.