Chlorophyll fluorescence is a common tool to investigate the behavior of the photosynthetic appratus, therefore photosynthetic capacity, at any physiological state. However, the different optical density of the samples can lead to light-dependent over- or underestimation of effective PSII quantum yields. This limitation can be overcome by the combination of multicolor light sheet microscopy and variable chlorophyll fluorescence imaging. This novel technique suggested by Lichtenberg et al. enables the calculation of depth-resolved photon absorption profiles to correct apparent PSII electron transport rates to photons absorbed by PSII, thus allows investigation of other photosynthetic systems with different anatomical structures and pigmentation as well as the role of specific accessory pigments in photosynthesis. (Summary by Quang Vuong Le) Plant Physiol. 10.1104/pp.17.00820.