The principles of the double-flash fluorometry
The sample chamber of the fluorometer PrimProd is illuminated with two xenon lamps, which produce actinic (pump) and probe flashes. During a weak probe flash (measuring light) chlorophyll fluorescence is measured with photosynthetic reaction centers of phytoplankton in the open (active) state; the fluorescence intensity reflects phytoplankton concentration in water samples.The actinic flash induces transition of reaction centers into the closed (inactive) state. If the probe flash is given after pump flash, before the closed centers have returned into the open state (i. e. the delay time between pump and probe flashes is comparable with the turnover time of reaction centers), then the intensity of the fluorescence exited by the probe flash will be higher than it was without previous actinic illumination. The increase in fluorescence yield is due to the inability of closed centers to use light energy for driving photosynthetic reactions.
Fig. Measuring chlorophyll fluorescence in PrimProd fluorometer.
Each measuring cycle involves a consequence of three flashes: probe-actinic-probe (see Fig). Chlorophyll fluorescence from open reaction centers (Fo) is measured during the first probe flash. Then, after the time interval selected by user, the actinic flash of saturating intensity follows (the minimum delay time is 1 s). The second probe flash is given 0.05 ms after the pump flash, when all the reaction centers still remain in the closed state. During the second probe flash, chlorophyll fluorescence from closed reaction centers (Fm) is measured.
Thus, Fo and Fm are determined in each cycle of measurement. Fo is used to calculate both phytoplankton concentration (by calibrating Fo versus chlorophyll or cells concentration, biomass) and light absorbtion capacity which is used for assessment the rate of algae photosynthesis. Photosynthetic activity (the efficiency of photochemical energy transformation in open reaction centers) is calculated as the ratio (Fm-Fo)/Fo. Variable fluorescence can be used to estimate photosynthetic rate and, accordingly, the primary production of phytoplankton.