Usually the initial measurement is the minimal level of fluorescence, . This is the fluorescence in the absence of photosynthetic light.
To use measurements of chlorophyll fluorescence to analyse photosynthesis, researchers must distinguish between photochemical quenching and non-photochemical quenching (heat dissipation). This is achieved by stopping photochemistry, which allows researchers to measure fluorescence in the presence of non-photochemical quenching alone. To reduce photochemical quenching to negligible levels, a high intensity, short flash of light is applied to the leaf. This transiently closes all PSII reaction centres, which prevents energy of PSII being passed to downstream electron carriers. Non-photochemical quenching will not be affected if the flash is short. During the flash, the fluorescence reaches the level reached in the absence of any photochemical quenching, known as maximum fluorescence .
The efficiency of photochemical quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing  to the steady yield of fluorescence in the light  and the yield of fluorescence in the absence of photosynthetic light .
The efficiency of non-photochemical quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in . Heat dissipation cannot be totally stopped, so the yield of chlorophyll fluorescence in the absence of non-photochemical quenching cannot be measured. Therefore, researchers use a dark-adapted point () with which to compare estimations of non-photochemical quenching.