Photoautotrophs environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here we describe a new approach for single-cell and subcellular photophysiology based on the customisation of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to: i. reassess the specialisation of photosynthetic activities in developing tissues of non-vascular plants; ii. identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which are consolidating metabolic connections with their animal hosts, and iii. testify to the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.

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