Pulse-Amplitude-Modulation (PAM)

Our PAM (Pulse-Amplitude-Modulation) chlorophyll fluorometer consists of a basic PAM101 system by Walz. The measuring light source gives 1 μsec pulses of 650 nm (pulsed LED, < 0.15 μmol photon m-2s-1). For actinic light, used to drive photosynthesis, also 650 nm light is used (LED, ~10-200 μmol photon m-2s-1), and saturating white light pulses are provided by a KL1500LCD (Schott; ~ 6000 μmol photon m-2s-1). A special holder for liquid samples (e.g. suspensions of leaf chloroplasts, microalgae, cyanobacteria and diatoms) is also available.

Photosynthesis involves reactions at five different functional levels

·         Processes at the pigment level

·         Primary light reactions

·         Thylakoid electron transport reactions

·         Dark-enzymatic stroma reactions

·         Slow regulatory feedback processes

In principle, chlorophyll fluorescence can function as an indicator at all of these levels of photosynthesis. Chlorophyll is the major antenna pigment, funneling the absorbed light energy into the reaction centers of photosystems I (PSI) and II (PSII), where photochemical conversion of the excitation energy takes place. The indicator function of chlorophyll fluorescence arises from the fact that fluorescence emission is complementary to the alternative pathways of de-excitation, which are photochemistry and heat dissipation. Generally speaking, the fluorescence yield is enhanced when the yields of photochemistry and heat dissipation are decreased and vice versa. The variable part of chlorophyll fluorescence originates mainly in PSII, thus reflecting changes in PSII photochemical efficiency and heat dissipation.

In practical applications, fluorometry in conjunction with the saturation pulse method has been particularly successful. The PAM fluorometry principle is based on a 1 μs pulse of light (low intensity, non-actinic) that is synchronized to a lock-in amplifier. This allows effective quantum yield determinations to be performed in (sun) light, as the lock-in amplifier removes all signal not associated with the lock-in signal. Following dark-adaptation, the minimum fluorescence value (Fo) of the sample can be measured. If the sample is then exposed to a saturating pulse of light, so all reaction centers have trapped the energy of a photon absorbed by their antennae (reaction centers are closed), the maximum amount of fluorescence (Fm) can be measured. The difference between these two extreme values is the variable fluorescence (Fv). Fv/Fm provides a measure of maximal PSII photochemical efficiency. Upon actinic illumination of the sample, its fluorescence yield can vary between the two extreme values, Fo and Fm. The momentary fluorescence yield, F, can be measured briefly before triggering of the saturating pulse, during which the maximal fluorescence yield, Fm’ is reached. The effective yield of PSII photochemical energy conversion under illumination is calculated as (Fm’-F)/Fm’.