We present a new system for microscopic multicolour variable chlorophyll fluorescence imaging of aquatic phototrophs. The system is compact and portable and enables microscopic imaging of photosynthetic performance of individual cells and chloroplasts using different combinations of blue, green, red or white light. Automated sequential exposure of microscopic samples to the three excitation colours enables subsequent deconvolution of the resulting fluorescence signals and colour marking of cells with different photopigmentation, i.e., cyanobacteria, green algae, red algae and diatoms. The photosynthetic activity in complex mixtures of phototrophs and natural samples can thus be assigned to different types of phototrophs, which can be quantified simultaneously. Here, we describe the composition and performance of the new imaging system and present applications with both natural phytoplankton and microalgal culture samples