Self-sustained luminescence enables non-invasive monitoring of molecular events at the whole-organism level, but its intensity depends on both gene expression and cellular metabolism, limiting quantitative interpretation. Here we present an approach that disentangles the contributions of gene expression and metabolism. By co-expressing an engineered reference luciferase variant that uses the same substrate but emits light at a different wavelength and monitoring the colour ratio, we substantially reduced signal variability, enabling quantitative physiological imaging despite changes in metabolic activity.