Often it is interesting to merge fluorescence and transmission images into one image because both sources have different information contents. Such an overlay image is generated by using the
HLS colour space as outlined in the following. The sources are a 24 bit
RGB image and an 8 bit monochrome image.
In a first step the RBG image is separated into its three HLS channels, see below. Note the reduced contrast of the lightness channel. As explained in Colour Images, the lightness gives the value of a pixel on the black-to-colour-to-white scale. In the autoscaled RGB image the brightest fluorescent areas have maximum intensity. This corresponds to neutral grey if the L channel is displayed in black and white as done below. Note further that the saturation channel is entirely white. The fluorescence colours are pure spectral colours without any grey content; they are maximally saturated.
Next, the L channel is contrast-optimized (or simply each value doubled) and designated as saturation channel while the greyscale transmission data are fed into lightness channel. Finally the three channels are recombined to an HSL image which gives the desired overlay.
The HSB colour space might also be used analogously for the overlay generation but the result looks less brilliant. The reason is that the most intensely fluorescent regions have maximal brightness (as compared to half maximal lightness) and consequently the channel cannot be contrast-enhanced before being used as saturation in the image recombination.
