Fluorescence-based methodologies are commonly employed to determine a wide spectrum of physiological parameters in intact photosynthetic organisms. These methods rely on the detection of Chlorophyll a fluorescent emission, which exhibits changes in its intensity due to the occurrence of quenching phenomena of either photochemical or non-photochemical nature, as well as in response of the absorption cross-section of the photosystems. At room temperature, it is generally considered that most of the emission stems from Photosystem II, and therefore, most of the physiological parameters rely on the assumption that contribution from Photosystem I is negligible. Moreover, it is often considered that the whole light-harvesting antenna is efficiently coupled to either of the photosystems and does not contribute, independently, to the detected emission. When these caveats are not realised, fluorescence-based indicators might be subjected to biases that tend to underestimate the extent of both photochemical and non-photochemical quenching. The contribution of Photosystem I and partially coupled/antenna components can be assessed through the analysis of the dependency of steady-state emission as a function of both the excitation and the emission wavelengths. On this basis, methods relying on using different combinations of excitation and emission wavelengths will be discussed in order to minimise the bias on the estimation of physiologically relevant parameters.